CMV glycoproteins and recombinant vectors

ABSTRACT

Disclosed herein are recombinant CMV vectors which may comprise a heterologous antigen that can repeatedly infect an organism while inducing a CD8+ T cell response to immunodominant epitopes of the heterologous antigen. The CMV vector may comprise a deleterious mutation in the US11 glycoprotein or a homolog thereof.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a divisional application of U.S. patent applicationSer. No. 14/086,602 filed Nov. 21, 2013, which issued as U.S. Pat. No.9,862,972 on Jan. 9, 2018. U.S. patent application Ser. No. 14/086,602is a continuation application of international patent application SerialNo. PCT/US12/41475 filed Jun. 8, 2012, which published as PCTPublication No. WO 2012/170765 on Dec. 13, 2012, which claims benefit ofand priority to U.S. provisional patent application Ser. No. 61/495,552,filed 10 Jun. 2011. Reference is made to international patentapplication Serial No. PCT/US11/29930 filed Mar. 25, 2011, U.S.provisional patent application Ser. No. 60/317,647 filed Mar. 25, 2010and U.S. patent application Ser. No. 11/597,457 filed Apr. 28, 2008.

FEDERAL FUNDING LEGEND

This invention was supported in part by the National Institutes ofHealth grant number ROI AI059457. The federal government may havecertain rights to this invention.

The foregoing applications, and all documents cited therein or duringtheir prosecution and all documents cited or referenced in theapplication, and all documents cited or referenced herein (“herein citeddocuments”), and all documents cited or referenced in herein citeddocuments, together with any manufacturer's instructions, descriptions,product specifications, and product sheets for any products mentionedherein or in any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. More specifically, all referenced documents areincorporated by reference to the same extent as if each individualdocument was specifically and individually indicated to be incorporatedby reference.

FIELD OF THE INVENTION

This invention relates to recombinant cytomegalovirus vectors, methodsof making them, uses for them, expression products from them, and usesthereof. This invention also relates to cytomegalovirus glycoproteinsUS2 to US11, in particular recombinant cytomegalovirus vectors lackingone or more of the glycoproteins US2 to US11, particularly US8 to US11,and more particularly, US11.

BACKGROUND OF THE INVENTION

HCMV is an ubiquitous virus that is present in over 60% of thepopulation depending on socioeconomic status. Following primaryinfection, HCMV persists for the life span of the host. Although HCMV isgenerally benign in healthy individuals, the virus can cause devastatingdisease in immunocompromised populations resulting in high morbidity andmortality (for review, see (Pass, R. F. 2001. Cytomegalovirus, p.2675-2705. In P. M. H. David M. Knipe, Diane E. Griffin, Robert A. Lamb,Malcolm A. Martin, Bernard Roizman and Stephen E. Straus (ed.), FieldsVirology, 4th ed. Lippincott Williams & Wilkins, Philadelphia,incorporated by reference herein).

CMV is one of the most immunogenic viruses known. High antibody titersare directed against numerous viral proteins during primary infection ofhealthy individuals (Alberola, J et al., J Clin Virol 16, 113-122(2000); Rasmussen L et al., J Infect Dis 164, 835-842 (1991); and(Farrell H E and Shellam G R, J Gen Virol 70 2573-2586 (1989), all ofwhich are incorporated by reference herein. In addition, a largeproportion of the host T cell repertoire is also directed against CMVantigens, with 5-10 fold higher median CD4+ T cell response frequenciesto HCMV than to acute viruses (measles, mumps, influenza, adenovirus) oreven other persistent viruses such as herpes simplex andvaricella-zoster viruses (Sylwester A W et al., J Exp Med 202, 673-685(2005). A high frequency of CD8+ responses to defined HCMV epitopes orproteins is also commonly observed (Gillespie G M et al., J Virol 74,8140-8150 (2000), Kern F et al., J Infect Dis 185, 1709-1716 (2002),Kern F et al., Eur J Immunol 29, 2908-2915 (1999), Kern F et al., JVirol 73, 8179-8184 (1999) and Sylwester A W et al., J Exp Med 202,673-685 (2005). In a large-scale human study quantifying CD4+ and CD8+ Tcell responses to the entire HCMV genome, the mean frequencies ofCMV-specific CD4+ and CD8+ T cells exceeded 10% of the memory populationfor both subsets and in some individuals, CMV-specific T cells toaccount for >25% of the memory T cell repertoire.

Paradoxically, the robust immune response to CMV is unable to eithereradicate the virus from healthy infected individuals or conferprotection against re-infection. This ability of CMV to escapeeradication by the immune system, and to re-infect the sero-positivehost has long been believed to be linked to the multiple viralimmunomodulators encoded by the virus (for review, see Mocarski E S etal., Trends Microbiol 10, 332-339 (2002) incorporated by referenceherein.) The HCMV US6 family of proteins (equivalent to RhCMVhomologues: Rh182-Rh189) are the most extensively studied of theseimmunomodulators (Loenen W A et al., Semin Immunol 13, 41-9 (2001);incorporated by reference herein.) At least four different genes, US2,US3, US6 and US11- and the respective RhCMV homologues (Rh182, Rh184,Rh185, and Rh189)—are known to interfere with assembly and transport ofMEC I molecules (Ahn K et al., Proc Natl Acad Sci USA 93, 10990-10995(1996), Ahn K et al., Immunity 6, 613-621 (1997.) Jones T R et al., JVirol 69, 4830-4841 (1995); Pantle N T et al., J Virol 79, 5786-5798,(2005). Wiertz E J et al., Cell 84, 769-779 (1996); and Wiertz E J etal., Nature 384, 432-438 (1996); all of which are incorporated byreference herein.)

Each of these four molecules interferes at different essential points ofMHC I protein maturation. US2 binds to newly synthesized MHC I heavychain (HC) and reverse translocates the protein through thetranslocation channel SEC61 back into the cytosol where HC is degradedby the proteasome. Similarly, US11 ejects MHC I back out into thecytoplasm. US3 and US6 act later in the MHC-I assembly process with US3retaining fully formed heterotrimers in the ER thus preventing theirtransport to the cell surface and US6 preventing peptide transport byTAP and thus formation of the trimeric complex of HC, β2m and peptide.

CMV-based vectors expressing heterologous antigens do not inducecytotoxic T cells directed against immunodominant epitopes of thoseheterologous antigens. This limits the efficacy of the T cells raised bya CMV-based vaccine to protect against infection by a pathogen or mounta cellular immune response against a tumor.

However, CMV-based vectors lacking viral inhibitors of antigenpresentation by MHC class I molecules—CMV based vectors that havedeleterious mutations in (including deletion of) all of US2, US3, US6,US8, US10, and US11 (US2-11 vectors) do indeed induce T cells to respondyo immunodominant antigens. (Hansen S G et al., Science 328, 102-106(2010). However, wild type US2, US3, US6, US8, US10, and US11 confersuperinfectivity in wild-type CMV vectors. Therefore vectors that havedeleterious mutations in all of US2, US3, US6, US8, US10, and US11 areeliminated by cytotoxic CD8+ T cells in individuals previouslyinoculated with CMV-vectors or naturally infected with CMV. Because thevast majority of humans have been exposed to CMV at some point in theirlives, CMV based vectors that have deleterious mutations in all of US2,US3, US6, US8, US10, and US11 would be of limited use.

The ability of wild type CMV to super-infect CMV-immune individuals andits inability to induce cytotoxic CD8+ T cells to immunodominantepitopes of heterologous antigens was thought to be intricately linked.Immunogenicity of CMV vectors was only be improved at the cost of losingthe ability to super-infect.

There is a need for CMV vectors that are able to super-infect CMV-immuneindividuals and induce an immune response, for example, cytotoxic CD8+ Tcells.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The present invention relates to viral vectors that overcome a crucialshortcoming in the development of vaccines based on cytomegalovirus(CMV).

The present invention relates to vectors that may have mutations (up toand including whole deletions) of the US8, US10, and US11 genes, butthat maintain functional homologues of US2, US3, and US6. These vectorsmay be useful in patients with prior CMV immunity, and generate acytotoxic T-cell response to immunodominant epitopes of heterologousantigens.

The present invention relates to HCMV vectors that have deleteriousmutations in, up to and including complete deletions of one or more HCMVglycoproteins. Such mutated glycoproteins include deleterious mutationsof one or more of US8, US10, or US11 (or functional homologues thereof)while leaving functional copies of US2-US6 (or functional homologuesthereof). In further examples, the HCMV vector may comprise adeleterious mutation, up to and including a complete deletion of US11,with functional copies of one or more of US2, US3, US6, US8, and US10remaining in the vector.

The present invention also relates to a method of generating an immuneresponse to a CMV heterologous antigen in a subject which may compriseadministering a CMV vector with a deleterious mutation in at least oneof US8, US10 or US11 or a functional homologue thereof and wherein theCMV vector contains and expresses a heterologous antigen. Theheterologous antigen may be any antigen, including pathogen-derived orcancer-derived antigens, including HIV antigens.

The applicants intend not to encompass within the invention anypreviously known product, process of making the product, or method ofusing the product such that Applicants reserve the right and herebydisclose a disclaimer of any previously known product, process, ormethod. It is further noted that the invention does not intend toencompass within the scope of the invention any product, process, ormaking of the product or method of using the product, which does notmeet the written description and enablement requirements of the USPTO(35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of the EPC),such that Applicants reserve the right and hereby disclose a disclaimerof any previously described product, process of making the product, ormethod of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1 depicts a set of two line graphs that compares CD8+ T cellepitope targeting of SIVgag-specific responses arising after vaccinationof Mamu A*0l+, CMV-naïve RM with wt vs. US2-11 knock-out (KO) RhCMV/gagvectors. The US2-11 KO vector elicits responses to all previouslycharacterized Mamu A*01-restricted gag epitopes, whereas wt CMV vectorselicit gag-specific CD8+ T cell responses that do not target theseepitopes (gag=total gag 15mer mixes).

FIG. 2 depicts a chart depicting the recognition of individual,consecutive gag 15mer peptides by 3 each Mamu A*01+, CMV-naïve RMvaccinated with wt vs. US2-11 knock-out (KO) RhCMV/gag vectors. Notethat whereas both wt and KO vectors elicit broad CD8+ T cell gag epitoperecognition, only the KO vector-elicited responses include recognitionof peptides containing conventional immunodominant epitopes (yellowrectangles; epitopes designated at top).

FIG. 3 depicts the RhCMV US2-11 region. MHC-I inhibitors are Rh182,Rh184, Rh185 and Rh189. Human CMV homologues are shown below.

FIGS. 4A-4B depict a diagram of viruses used in Example 2. Regions ofthe genome that were altered to create mutant viruses are shown here indetail. All RhCMV ORFs are depicted as arrows that correspond to thedirection of the ORF within the genome. Blue arrows represent genes thatdownregulate MHC class I. Designated RhCMV nomenclature is used for allORFs. For ORFs with homology to HCMV genes the name of the correspondingHCMV homologue is shown in brackets. Also depicted are SIV immunologicalmarkers SIVgag and RTN, and recombination sites LoxP, FRT, and F5 FRT.

FIG. 5A depicts the characterization of recombinant RhCMVs by RT-PCR.Fibroblasts were infected at MOI=1 with the indicated virus and totalRNA was harvested at 24hpi (Δ6-9gag=ΔUS8-11gag). cDNA was synthesized byrandom hexamer priming, and transcripts were amplified with primersspecific for the ORFs indicated on the left. Genes flanking the deletedregions were included to detect possible changes in transcription due tothe deletions. WT=bacterial artificial chromosome (BAC)-derived wildtype RhCMV. RT=reverse transcriptase.

FIG. 5B depicts the expression of SIVgag and SIV RTN by recombinantviruses. Immunoblot analysis of FLAG-tagged SIVgag and V5-tagged SIV RTNwas performed at the indicated times after fibroblasts were infected atMOI=1 and total lysate was harvested.

FIG. 6A depicts the boosted RhCMV-specific CD4+ T cell response in PBMCand BAL. Boosting of pre-existing anti-CMV T cell responses are a signof super-infection by the incoming vector.

FIG. 6B depicts the development of total SIVgag-specific CD4+ and CD8+ Tcell response in PBMC and BAL. The development of a de novo SIVgagresponse is proof for super-infection.

FIG. 6C depicts the development of CD8+ T cell response in PBMC tospecific SIVgag-derived peptides that are known Mamu A*01-restrictedepitopes. The development of T cell responses against immunodominantepitopes is in contrast to the lack of these responses uponsuper-infection with wild-type RhCMV expressing gag (FIG. 1).

FIG. 7A is a line graph depicting the percentage of cells in the blood(left) and BAL (right) responding to SIVrtn and SIVgag in RM inoculatedwith ΔUS8-11RhCMV/rtn and ΔUS8-11RhCMV/gag vectors over time postinoculation.

FIG. 7B is a line graph depicting the percentage of cells in the blood(left) and BAL (right) responding to the immunodominant MamuA*01-restricted epitopes SIVtat(SL8) and SIVgag(CM9) determined by flowcytometric analysis in RM inoculated with ΔUS8-11RhCMV/rtn andΔUS8-11RhCMV/gag vectors over time post inoculation.

FIG. 8A is a line graph depicting the percentage of cells in the blood(left) and BAL (right) responding to SIVrtn and SIVgag in RM inoculatedwith ΔUS2-6RhCMV/rtn and ΔUS2-6RhCMV/gag vectors over time postinoculation.

FIG. 8B is a line graph depicting the percentage of cells in the blood(left) and BAL (right) responding to the immunodominant MamuA*01-restricted epitopes SIVtat(SL8) and SIVgag(CM9) determined by flowcytometric analysis in RM inoculated with ΔUS2-6RhCMV/rtn andΔUS2-6RhCMV/gag vectors over time post inoculation. No responding cellswere detected.

FIG. 9A is a schematic representation of the construct RTNΔ189gag.

FIG. 9B is an image of a gel that shows the results of PCR amplificationof the constructs of FIG. 9A verifying Rh189-deletion and SIVgaginsertion.

FIG. 9C is an image of an immunoblot probing for SIVretanef in theindicated constructs.

FIG. 10 is a flow diagram of cells responding to RTN and itsimmunodominant peptide SL8-tat in a rhesus macaque inoculated withRhCMV/RTNΔ189gag, showing that a deleterious mutation in US11 alone issufficient to confer superinfectivity and presentation of immunodominantepitopes.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a CMV vector capable of repeatedly infecting anorganism which may comprise a deleterious mutation in the glycoproteinUS11 of such a character that the mutation renders the particularglycoprotein non-functional or causes a reduction in function. Themutation may be any mutation, including a point mutation, a frameshiftmutation, and a deletion of less than all of the glycoprotein, thedeletion of the entire glycoprotein, or the deletion of the nucleic acidsequence encompassing all of US8, US10, and US11 and all interveningsequences. In further examples, the CMV vector may comprise adeleterious mutation in US11, up to and including the deletion of all ofthe US11 ORF.

For example, FIGS. 6A, 6B and 6C show that a viral vector with adeletion of US8-11 is still capable of superinfection of CMV-positiveanimals and that CMV lacking US8-11 induces a T cell response toimmunodominant SIV epitopes. Two CMV-positive rhesus macaques (RM)(#26597 & #27198) were inoculated subcutaneously with 10⁷ PFU ofrecombinant US8-llgag. Responses frequencies were determined by flowcytometric analysis of intracellular cytokine staining for CD69, TNF-αand interferon-γ using RhCMV or overlapping 15mer peptides correspondingto SIVgag. The percentage of the responding, SIVgag specific T cellswithin the overall memory subset is shown for each time point.RhCMV-specific responses were measured by adding purified virus.

The mutations may be random or site-directed. For random mutations,mutagenic agents, in particular alkylating mutagenic agents, are diethylsulfate (des), ethyleneimine (ei), propane sultone,N-methyl-N-nitrosourethane (mnu), N-nitroso-N-methylurea (NMU),N-ethyl-N-nitrosourea (enu), sodium azide may be utilized.Alternatively, the mutations may be induced by means of irradiation,which is for example selected from x-rays, fast neutrons, UVirradiation.

Mutations may be introduced using synthetic oligonucleotides. Theseoligonucleotides contain nucleotide sequences flanking the desiredmutation sites. A suitable method is disclosed in Morinaga et al.(Biotechnology (1984)2, p 646-649). Another method of introducingmutations into enzyme-encoding nucleotide sequences is described inNelson and Long (Analytical Biochemistry (1989), 180, p 147-151).Instead of site directed mutagenesis, such as described above, one canintroduce mutations randomly for instance using a commercial kit such asthe GeneMorph PCR mutagenesis kit from Stratagene, or the Diversify PCRrandom mutagenesis kit from Clontech. EP 0 583 265 refers to methods ofoptimising PCR based mutagenesis, which can also be combined with theuse of mutagenic DNA analogues such as those described in EP 0 866 796.Error prone PCR technologies are suitable for the production of variantsof lipid acyl transferases with preferred characteristics.

Antisense techniques as well as direct gene manipulation are known foruse in modulating gene expression. The invention thus includes the useof antisense nucleic acids, which may incorporate natural or modifiednucleotides, or both, ribozymes, including hammerhead ribozymes, geneknockout such as by homologous recombination, and other techniques forreducing gene expression levels.

RNA interference (RNAi) is a method of post transcriptional genesilencing (PTGS) induced by the direct introduction of double-strandedRNA (dsRNA) and has emerged as a useful tool to knock out expression ofspecific genes in a variety of organisms. RNAi is described by Fire etal., Nature 391:806-811 (1998). Other methods of PTGS are known andinclude, for example, introduction of a transgene or virus. Generally,in PTGS, the transcript of the silenced gene is synthesised but does notaccumulate because it is rapidly degraded. Methods for PTGS, includingRNAi are described, for example, in the Ambion.com world wide web site,in the directory “/hottopics/”, in the “mai” file. Suitable methods forRNAi in vitro are known to those skilled in the art. One such methodinvolves the introduction of siRNA (small interfering RNA). Currentmodels indicate that these 21-23 nucleotide dsRNAs can induce PTGS.Methods for designing effective siRNAs are described, for example, inthe Ambion web site described above.

CMV vectors, when used as expression vectors are innately non-pathogenicin the selected subjects such as humans or have been modified to renderthem non-pathogenic in the selected subjects. For example,replication-defective adenoviruses and alphaviruses are well known andcan be used as gene delivery vectors. Without US2-11 all of thesevectors (except for CMV which contains US2-11 naturally) elicitvector-specific immunity which prohibits their repeated use.

The present invention also relates to a method of inducing a CD8+ T cellresponse in a subject, which may comprise (a) administering a CMV vectorwith at least one cytomegalovirus (CMV) glycoprotein deleted from theCMV vector, wherein the glycoprotein is US11, and wherein the CMV vectorcontains and expresses at least one heterologous (non-CMV) antigen and(b) administering the vector to the animal or human subject.

The heterologous antigen may be derived from a pathogen. The pathogenmay be a viral pathogen and the antigen may be a protein derived fromthe viral pathogen. Viruses include, but are not limited to Adenovirus,coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, Herpessimplex, type 1, Herpes simplex, type 2, Varicella-zoster virus,Epstein-barr virus, Kaposi's sarcoma herpesvirus, Human cytomegalovirus,Human herpesvirus, type 8, Hepatitis B virus, Hepatitis C virus, yellowfever virus, dengue virus, West Nile virus, Human immunodeficiency virus(HIV), Influenza virus, Measles virus, Mumps virus, Parainfluenza virus,Respiratory syncytial virus, Human metapneumovirus, Humanpapillomavirus, Rabies virus, Rubella virus, Human bocavirus andParvovirus B19.

The pathogen may be a bacterial pathogen and the antigen may be aprotein derived from the bacterial pathogen. The pathogenic bacteriainclude, but are not limited to, Bordetella pertussis, Borreliaburgdoiferi, Brucella abortus, Brucella canis, Brucella melitensis,Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydiatrachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridiumdifficile, Clostridium perfringens, Clostridium tetani, Corynebacteriumdiphtherias, Enterococcus faecalis, Enterococcus faecium, Escherichiacoli, Francisella tularensis, Haemophilus influenzae, Helicobacterpylori, Legionella pneumophila, Leptospira interrogans, Listeriamonocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis,Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae,Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii,Salmonella typhi, Salmonella typhimurium, Shigella sonnei,Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcussaprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae,Streptococcus pyogenes, Treponema pallidum, Vibrio cholera and Yersiniapestis.

The pathogen may be a parasite and the antigen may be a protein derivedfrom the parasite pathogen. The parasite may be a protozoan organism ordisease caused by a protozoan organism such as, but not limited to,Acanthamoeba, Babesiosis, Balantidiasis, Blastocystosis, Coccidia,Dientamoebiasis, Amoebiasis, Giardia, Isosporiasis, Leishmaniasis,Primary amoebic meningoencephalitis (PAM), Malaria, Rhinosporidiosis,Toxoplasmosis—Parasitic pneumonia, Trichomoniasis, Sleeping sickness andChagas disease. The parasite may be a helminth organism or worm or adisease caused by a helminth organism such as, but not limited to,Ancylostomiasis/Hookworm, Anisakiasis, Roundworm—Parasitic pneumonia,Roundworm—Baylisascariasis, Tapeworm—Tapeworm infection, Clonorchiasis,Dioctophyme renalis infection, Diphyllobothriasis—tapeworm, Guineaworm—Dracunculiasis, Echinococcosis—tapeworm, Pinworm—Enterobiasis,Liver fluke—Fasciolosis, Fasciolopsiasis—intestinal fluke,Gnathostomiasis, Hymenolepiasis, Loa loa filariasis, Calabar swellings,Mansonelliasis, Filariasis, Metagonimiasis—intestinal fluke, Riverblindness, Chinese Liver Fluke, Paragonimiasis, Lung Fluke,Schistosomiasis—bilharzia, bilharziosis or snail fever (all types),intestinal schistosomiasis, urinary schistosomiasis, Schistosomiasis bySchistosoma japonicum, Asian intestinal schistosomiasis, Sparganosis,Strongyloidiasis—Parasitic pneumonia, Beef tapeworm, Pork tapeworm,Toxocariasis, Trichinosis, Swimmer's itch, Whipworm and ElephantiasisLymphatic filariasis. The parasite may be an organism or disease causedby an organism such as, but not limited to, parasitic worm, HalzounSyndrome, Myiasis, Chigoe flea, Human Botfly and Candiru. The parasitemay be an ectoparasite or disease caused by an ectoparasite such as, butnot limited to, Bedbug, Head louse—Pediculosis, Body louse—Pediculosis,Crab louse—Pediculosis, Demodex-Demodicosis, Scabies, Screwworm andCochliomyia.

The antigen may be a protein derived from cancer. The cancers, include,but are not limited to, Acute lymphoblastic leukemia; Acute myeloidleukemia; Adrenocortical carcinoma; AIDS-related cancers; AIDS-relatedlymphoma; Anal cancer; Appendix cancer; Astrocytoma, childhoodcerebellar or cerebral; Basal cell carcinoma; Bile duct cancer,extrahepatic; Bladder cancer; Bone cancer, Osteosarcoma/Malignantfibrous histiocytoma; Brainstem glioma; Brain tumor; Brain tumor,cerebellar astrocytoma; Brain tumor, cerebral astrocytoma/malignantglioma; Brain tumor, ependymoma; Brain tumor, medulloblastoma; Braintumor, supratentorial primitive neuroectodermal tumors; Brain tumor,visual pathway and hypothalamic glioma; Breast cancer; Bronchialadenomas/carcinoids; Burkitt lymphoma; Carcinoid tumor, childhood;Carcinoid tumor, gastrointestinal; Carcinoma of unknown primary; Centralnervous system lymphoma, primary; Cerebellar astrocytoma, childhood;Cerebral astrocytoma/Malignant glioma, childhood; Cervical cancer;Childhood cancers; Chronic lymphocytic leukemia; Chronic myelogenousleukemia; Chronic myeloproliferative disorders; Colon Cancer; CutaneousT-cell lymphoma; Desmoplastic small round cell tumor; Endometrialcancer; Ependymoma; Esophageal cancer; Ewing's sarcoma in the Ewingfamily of tumors; Extracranial germ cell tumor, Childhood; ExtragonadalGerm cell tumor; Extrahepatic bile duct cancer; Eye Cancer, Intraocularmelanoma; Eye Cancer, Retinoblastoma; Gallbladder cancer; Gastric(Stomach) cancer; Gastrointestinal Carcinoid Tumor; Gastrointestinalstromal tumor (GIST); Germ cell tumor: extracranial, extragonadal, orovarian; Gestational trophoblastic tumor; Glioma of the brain stem;Glioma, Childhood Cerebral Astrocytoma; Glioma, Childhood Visual Pathwayand Hypothalamic; Gastric carcinoid; Hairy cell leukemia; Head and neckcancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin lymphoma;Hypopharyngeal cancer; Hypothalamic and visual pathway glioma,childhood; Intraocular Melanoma; Islet Cell Carcinoma (EndocrinePancreas); Kaposi sarcoma; Kidney cancer (renal cell cancer); LaryngealCancer; Leukemias; Leukemia, acute lymphoblastic (also called acutelymphocytic leukemia); Leukemia, acute myeloid (also called acutemyelogenous leukemia); Leukemia, chronic lymphocytic (also calledchronic lymphocytic leukemia); Leukemia, chronic myelogenous (alsocalled chronic myeloid leukemia); Leukemia, hairy cell; Lip and OralCavity Cancer; Liver Cancer (Primary); Lung Cancer, Non-Small Cell; LungCancer, Small Cell; Lymphomas; Lymphoma, AIDS-related; Lymphoma,Burkitt; Lymphoma, cutaneous T-Cell; Lymphoma, Hodgkin; Lymphomas,Non-Hodgkin (an old classification of all lymphomas except Hodgkin's);Lymphoma, Primary Central Nervous System; Marcus Whittle, DeadlyDisease; Macroglobulinemia, Waldenström; Malignant Fibrous Histiocytomaof Bone/Osteosarcoma; Medulloblastoma, Childhood; Melanoma; Melanoma,Intraocular (Eye); Merkel Cell Carcinoma; Mesothelioma, Adult Malignant;Mesothelioma, Childhood; Metastatic Squamous Neck Cancer with OccultPrimary; Mouth Cancer; Multiple Endocrine Neoplasia Syndrome, Childhood;Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides;Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Diseases;Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; MyeloidLeukemia, Childhood Acute; Myeloma, Multiple (Cancer of theBone-Marrow); Myeloproliferative Disorders, Chronic; Nasal cavity andparanasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma;Non-Hodgkin lymphoma; Non-small cell lung cancer; Oral Cancer;Oropharyngeal cancer; Osteosarcoma/malignant fibrous histiocytoma ofbone; Ovarian cancer; Ovarian epithelial cancer (Surfaceepithelial-stromal tumor); Ovarian germ cell tumor; Ovarian lowmalignant potential tumor; Pancreatic cancer; Pancreatic cancer, isletcell; Paranasal sinus and nasal cavity cancer; Parathyroid cancer;Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal astrocytoma;Pineal germinoma; Pineoblastoma and supratentorial primitiveneuroectodermal tumors, childhood; Pituitary adenoma; Plasma cellneoplasia/Multiple myeloma; Pleuropulmonary blastoma; Primary centralnervous system lymphoma; Prostate cancer; Rectal cancer; Renal cellcarcinoma (kidney cancer); Renal pelvis and ureter, transitional cellcancer; Retinoblastoma; Rhabdomyosarcoma, childhood; Salivary glandcancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, softtissue; Sarcoma, uterine; Sezary syndrome; Skin cancer (nonmelanoma);Skin cancer (melanoma); Skin carcinoma, Merkel cell; Small cell lungcancer; Small intestine cancer; Soft tissue sarcoma; Squamous cellcarcinoma—see Skin cancer (nonmelanoma); Squamous neck cancer withoccult primary, metastatic; Stomach cancer; Supratentorial primitiveneuroectodermal tumor, childhood; T-Cell lymphoma, cutaneous (MycosisFungoides and Sezary syndrome); Testicular cancer; Throat cancer;Thymoma, childhood; Thymoma and Thymic carcinoma; Thyroid cancer;Thyroid cancer, childhood; Transitional cell cancer of the renal pelvisand ureter; Trophoblastic tumor, gestational; Unknown primary site,carcinoma of, adult; Unknown primary site, cancer of, childhood; Ureterand renal pelvis, transitional cell cancer; Urethral cancer; Uterinecancer, endometrial; Uterine sarcoma; Vaginal cancer; Visual pathway andhypothalamic glioma, childhood; Vulvar cancer; Waldenstr6mmacroglobulinemia and Wilms tumor (kidney cancer).

Accordingly, the invention provides a CMV synthetically modified tocontain therein exogenous DNA. The CMV has had US11 deleted therefrom.

The invention further provides a vector for cloning or expression ofheterologous DNA which may comprise the recombinant CMV.

The heterologous DNA may encode an expression product which maycomprise: an epitope of interest, a biological response modulator, agrowth factor, a recognition sequence, a therapeutic gene, or a fusionprotein.

An epitope of interest is an antigen or immunologically active fragmentthereof from a pathogen or toxin of veterinary or human interest.

An epitope of interest can be an antigen of pathogen or toxin, or froman antigen of a pathogen or toxin, or another antigen or toxin whichelicits a response with respect to the pathogen, or from another antigenor toxin which elicits a response with respect to the pathogen.

An epitope of interest can be an antigen of a human pathogen or toxin,or from an antigen of a human pathogen or toxin, or another antigen ortoxin which elicits a response with respect to the pathogen, or fromanother antigen or toxin which elicits a response with respect to thepathogen, such as, for instance: a Morbillivirus antigen, e.g., ameasles virus antigen such as HA or F; a rabies glycoprotein, e.g.,rabies virus glycoprotein G; an influenza antigen, e.g., influenza virusHA or N; a Herpesvirus antigen, e.g., a glycoprotein of a herpes simplexvirus (HSV), a human cytomegalovirus (HCMV), Epstein-Barr; a flavivirusantigen, a JEV, Yellow Fever virus or Dengue virus antigen; a Hepatitisvirus antigen, e.g., HBsAg; an immunodeficiency virus antigen, e.g., anHIV antigen such as gp120, gp160; a Hantaan virus antigen; a C. tetaniantigen; a mumps antigen; a pneumococcal antigen, e.g., PspA; a Borreliaantigen, e.g., OspA, OspB, OspC of Borrelia associated with Lyme diseasesuch as Borrelia burgdorferi, Borrelia atzelli and Borrelia garinii; achicken pox (varicella zoster) antigen; or a Plasmodium antigen.

The epitope of interest may be derived from an antigen of animmunodeficiency virus such as HIV or SIV. However, the epitope ofinterest can be an antigen of any veterinary or human pathogen or fromany antigen of any veterinary or human pathogen.

Since the heterologous DNA can encode a growth factor or therapeuticgene, the recombinant CMV can be used in gene therapy. Gene therapyinvolves transferring genetic information; and, with respect to genetherapy and immunotherapy, reference is made to U.S. Pat. No. 5,252,479,which is incorporated herein by reference, together with the documentscited in it and on its face, and to WO 94/16716 and U.S. applicationSer. No. 08/184,009, filed Jan. 19, 1994, each of which is alsoincorporated herein by reference, together with the documents citedtherein. The growth factor or therapeutic gene, for example, can encodea disease-fighting protein, a molecule for treating cancer, a tumorsuppressor, a cytokine, a tumor associated antigen, or interferon; and,the growth factor or therapeutic gene can, for example, be selected fromthe group consisting of a gene encoding alpha-globin, beta-globin,gamma-globin, granulocyte macrophage-colony stimulating factor, tumornecrosis factor, an interleukin, macrophage colony stimulating factor,granulocyte colony stimulating factor, erythropoietin, mast cell growthfactor, tumor suppressor p53, retinoblastoma, interferon, melanomaassociated antigen or B7.

The invention still further provides an immunogenic, immunological orvaccine composition containing the recombinant CMV virus or vector, anda pharmaceutically acceptable carrier or diluent. An immunologicalcomposition containing the recombinant CMV virus or vector (or anexpression product thereof) elicits an immunological response—local orsystemic. The response can, but need not be, protective. An immunogeniccomposition containing the recombinant CMV virus or vector (or anexpression product thereof) likewise elicits a local or systemicimmunological response which can, but need not be, protective. A vaccinecomposition elicits a local or systemic protective response.Accordingly, the terms “immunological composition” and “immunogeniccomposition” include a “vaccine composition” (as the two former termscan be protective compositions).

The invention therefore also provides a method of inducing animmunological response in a host vertebrate which may compriseadministering to the host an immunogenic, immunological or vaccinecomposition which may comprise the recombinant CMV virus or vector and apharmaceutically acceptable carrier or diluent. For purposes of thisspecification, the term “subject” includes all animals and humans, while“animal” includes all vertebrate species, except humans; and“vertebrate” includes all vertebrates, including animals (as “animal” isused herein) and humans. And, of course, a subset of “animal” is“mammal”, which for purposes of this specification includes all mammals,except humans.

The invention even further provides a therapeutic composition containingthe recombinant CMV virus or vector and a pharmaceutically acceptablecarrier or diluent. The therapeutic composition is useful in the genetherapy and immunotherapy embodiments of the invention, e.g., in amethod for transferring genetic information to an animal or human inneed of such which may comprise administering to the host thecomposition; and, the invention accordingly includes methods fortransferring genetic information.

In yet another embodiment, the invention provides a method of expressinga protein or gene product or an expression product which may compriseinfecting or transfecting a cell in vitro with a recombinant CMV virusor vector of the invention and optionally extracting, purifying orisolating the protein, gene product or expression product or DNA fromthe cell. And, the invention provides a method for cloning orreplicating a heterologous DNA sequence which may comprise infecting ortransfecting a cell in vitro or in vivo with a recombinant CMV virus orvector of the invention and optionally extracting, purifying orisolating the DNA from the cell or progeny virus.

The invention in another aspect provides a method for preparing therecombinant CMV virus or vector of the invention which may compriseinserting the exogenous DNA into a non-essential region of the CMVgenome.

The method can further comprise deleting a non-essential region from theCMV genome, preferably prior to inserting the exogenous DNA.

The method can comprise in vivo recombination. Thus, the method cancomprise transfecting a cell with CMV DNA in a cell-compatible medium inthe presence of donor DNA which may comprise the exogenous DNA flankedby DNA sequences homologous with portions of the CMV genome, whereby theexogenous DNA is introduced into the genome of the CMV, and optionallythen recovering CMV modified by the in vivo recombination.

The method can also comprise cleaving CMV DNA to obtain cleaved CMV DNA,ligating the exogenous DNA to the cleaved CMV DNA to obtain hybridCMV-exogenous DNA, transfecting a cell with the hybrid CMV-exogenousDNA, and optionally then recovering CMV modified by the presence of theexogenous DNA.

Since in vivo recombination is comprehended, the invention accordinglyalso provides a plasmid which may comprise donor DNA not naturallyoccurring in CMV encoding a polypeptide foreign to CMV, the donor DNA iswithin a segment of CMV DNA which would otherwise be co-linear with anon-essential region of the CMV genome such that DNA from anon-essential region of CMV is flanking the donor DNA.

The exogenous DNA can be inserted into CMV to generate the recombinantCMV in any orientation which yields stable integration of that DNA, andexpression thereof, when desired.

The exogenous DNA in the recombinant CMV virus or vector of theinvention can include a promoter. The promoter can be from a herpesvirus. For instance, the promoter can be a cytomegalovirus (CMV)promoter, such as a human CMV (HCMV) or murine CMV promoter. Thepromoter can also be a non-viral promoter such as the EF1α promoter.

The promoter may be a truncated transcriptionally active promoter whichmay comprise a region transactivated with a transactivating proteinprovided by the virus and the minimal promoter region of the full-lengthpromoter from which the truncated transcriptionally active promoter isderived. For purposes of this specification, a “promoter” is composed ofan association of DNA sequences corresponding to the minimal promoterand upstream regulatory sequences; a “minimal promoter” is composed ofthe CAP site plus TATA box (minimum sequences for basic level oftranscription; unregulated level of transcription); and, “upstreamregulatory sequences” are composed of the upstream element(s) andenhancer sequence(s). Further, the term “truncated” indicates that thefull-length promoter is not completely present, i.e., that some portionof the full-length promoter has been removed. And, the truncatedpromoter can be derived from a herpesvirus such as MCMV or HCMV, e.g.,HCMV-IE or MCMV-IE.

Like the aforementioned promoter, the inventive promoter can be aherpesvirus, e.g., a MCMV or HCMV such as MCMV-IE or HCMV-IE promoter;and, there can be up to a 40% and even up to a 90% reduction in size,from a full-length promoter, based upon base pairs. The promoter canalso be a modified non-viral promoter.

The invention thus also provides an expression cassette for insertioninto a recombinant virus or plasmid which may comprise the truncatedtranscriptionally active promoter. The expression cassette can furtherinclude a functional truncated polyadenylation signal; for instance anSV40 polyadenylation signal which is truncated, yet functional.Considering that nature provided a larger signal, it is indeedsurprising that a truncated polyadenylation signal is functional; and, atruncated polyadenylation signal addresses the insert size limitproblems of recombinant viruses such as CMV. The expression cassette canalso include exogenous or heterologous DNA with respect to the virus orsystem into which it is inserted; and that DNA can be exogenous orheterologous DNA as described herein.

In a more specific aspect, the present invention encompasses CMV,recombinants which may comprise viral or non-viral promoters, preferablya truncated promoter therefrom. The invention further comprehendsantibodies elicited by the inventive compositions and/or recombinantsand uses for such antibodies. The antibodies, or the product (epitopesof interest) which elicited them, or monoclonal antibodies from theantibodies, can be used in binding assays, tests or kits to determinethe presence or absence of an antigen or antibody.

Flanking DNA used in the invention can be from the site of insertion ora portion of the genome adjacent thereto (wherein “adjacent” includescontiguous sequences, e.g., codon or codons, as well as up to as manysequences, e.g., codon or codons, before there is an interveninginsertion site).

The exogenous or heterologous DNA (or DNA foreign to CMV, or DNA notnaturally occurring in CMV) can be DNA encoding any of theaforementioned epitopes of interest, as listed above. The exogenous DNAcan include a marker, e.g., a color or light marker. The exogenous DNAcan also code for a product which would be detrimental to an insect hostsuch that the expression product can be a pesticide or insecticide. Theexogenous DNA can also code for an anti-fungal polypeptide; and, forinformation on such a polypeptide and DNA therefor, reference is made toU.S. Pat. No. 5,421,839 and the documents cited therein, incorporatedherein by reference.

The heterologous or exogenous DNA in recombinants of the inventionpreferably encodes an expression product which may comprise: an epitopeof interest, a biological response modulator, a growth factor, arecognition sequence, a therapeutic gene, or a fusion protein. Withrespect to these terms, reference is made to the following discussion,and generally to Kendrew, THE ENCYCLOPEDIA OF MOLECULAR BIOLOGY(Blackwell Science Ltd 1995) and Sambrook, Fritsch, Maniatis, MolecularCloning, A LABORATORY MANUAL (2d Edition, Cold Spring Harbor LaboratoryPress, 1989).

As to antigens for use in vaccine or immunological compositions, seealso Stedman's Medical Dictionary (24th edition, 1982), e.g., definitionof vaccine (for a list of antigens used in vaccine formulations; suchantigens or epitopes of interest from those antigens can be used in theinvention, as either an expression product of the inventive recombinantvirus, or in a multivalent composition containing an inventiverecombinant virus or an expression product therefrom).

As to epitopes of interest, one skilled in the art can determine anepitope or immunodominant region of a peptide or polypeptide and ergothe coding DNA therefor from the knowledge of the amino acid andcorresponding DNA sequences of the peptide or polypeptide, as well asfrom the nature of particular amino acids (e.g., size, charge, etc.) andthe codon dictionary, without undue experimentation.

A general method for determining which portions of a protein to use m animmunological composition focuses on the size and sequence of theantigen of interest. “In general, large proteins, because they have morepotential determinants are better antigens than small ones. The moreforeign an antigen, that is the less similar to self-configurationswhich induce tolerance, the more effective it is in provoking an immuneresponse.” Ivan Roitt, Essential Immunology, 1988.

As to size: the skilled artisan can maximize the size of the proteinencoded by the DNA sequence to be inserted into the viral vector(keeping in mind the packaging limitations of the vector). To minimizethe DNA inserted while maximizing the size of the protein expressed, theDNA sequence can exclude introns (regions of a gene which aretranscribed but which are subsequently excised from the primary RNAtranscript).

At a minimum, the DNA sequence can code for a peptide at least 8 or 9amino acids long. This is the minimum length that a peptide needs to bein order to stimulate a CD8+ T cell response (which recognizes virusinfected cells or cancerous cells). A minimum peptide length of 13 to 25amino acids is useful to stimulate a CD4+ T cell response (whichrecognizes special antigen presenting cells which have engulfed thepathogen). See Kendrew, supra. However, as these are minimum lengths,these peptides are likely to generate an immunological response, i.e.,an antibody or T cell response; but, for a protective response (as froma vaccine composition), a longer peptide is preferred.

With respect to the sequence, the DNA sequence preferably encodes atleast regions of the peptide that generate an antibody response or a Tcell response. One method to determine T and B cell epitopes involvesepitope mapping. The protein of interest “is fragmented into overlappingpeptides with proteolytic enzymes or overlapping peptides are generatedby oligo-peptide synthesis. The individual peptides are then tested fortheir ability to bind to an antibody elicited by the native protein orto induce T cell or B cell activation. This approach has beenparticularly useful in mapping T-cell epitopes since the T cellrecognizes short linear peptides complexed with MEC molecules (see FIG.2). The method is less effective for determining B-cell epitopes” sinceB cell epitopes are often not linear amino acid sequences but ratherresult from the tertiary structure of the folded three dimensionalprotein. Janis Kuby, Immunology, (1992) pp. 79-80.

Another method of determining an epitope of interest is to choose theregions of the protein that are hydrophilic. Hydrophilic residues areoften on the surface of the protein and are therefore often the regionsof the protein which are accessible to the antibody. Janis Kuby,Immunology, (1992) p. 81.

Yet another method for determining an epitope of interest is to performan X-ray crystallographic analysis of the antigen (full length)-antibodycomplex. Janis Kuby, Immunology, (1992) p. 80.

Still another method for choosing an epitope of interest which cangenerate a T cell response is to identify from the protein sequencepotential HLA anchor binding motifs which are peptide sequences whichare known to be likely to bind to the MHC molecule.

The peptide which is a putative epitope of interest, to generate a Tcell response, should be presented in a MHC complex. The peptidepreferably contains appropriate anchor motifs for binding to the MHCmolecules, and should bind with high enough affinity to generate animmune response. Factors which can be considered are: the HLA type ofthe patient (vertebrate, animal or human) expected to be immunized, thesequence of the protein, the presence of appropriate anchor motifs andthe occurrence of the peptide sequence in other vital cells.

An immune response is generated, in general, as follows: T cellsrecognize proteins only when the protein has been cleaved into smallerpeptides and is presented in a complex called the “majorhistocompatibility complex (MHC)” located on another cell's surface.There are two classes of MHC complexes—class I and class II, and eachclass is made up of many different alleles. Different species, andindividual subjects have different types of MEC complex alleles; theyare said to have a different HLA type.

Class I MHC complexes are found on virtually every cell and presentpeptides from proteins produced inside the cell. Thus, Class I MHCcomplexes are useful for killing cells which when infected by viruses orwhich have become cancerous and as the result of expression of anoncogene. T cells which have a protein called CD8 on their surface, bindto the MHC class I cells and secrete lymphokines. The lymphokinesstimulate a response; cells arrive and kill the viral infected cell.

Class II MHC complexes are found only on antigen-presenting cells andare used to present peptides from circulating pathogens which have beenendocytosed by the antigen-presenting cells. T cells which have aprotein called CD4 bind to the MHC class II cells and kill the cell byexocytosis of lytic granules.

Some guidelines in determining whether a protein is an epitope ofinterest which will stimulate a T cell response, include: Peptidelength—the peptide should be at least 8 or 9 amino acids long to fitinto the MHC class I complex and at least 13-25 amino acids long to fitinto a class II MCH complex. This length is a minimum for the peptide tobind to the MHC complex. It is preferred for the peptides to be longerthan these lengths because cells may cut the expressed peptides. Thepeptide should contain an appropriate anchor motif which will enable itto bind to the various class I or class II molecules with high enoughspecificity to generate an immune response (See Bocchia, M. et al.,Specific Binding of Leukemia Oncogene Fusion Protein Peptides to HLAClass I Molecules, Blood 85:2680-2684; Englehard, V H, Structure ofpeptides associated with class I and class II MEC molecules, Ann. Rev.Immunol. 12:181 (1994)). This can be done, without undueexperimentation, by comparing the sequence of the protein of interestwith published structures of peptides associated with the MHC molecules.Protein epitopes recognized by T cell receptors are peptides generatedby enzymatic degradation of the protein molecule and are presented onthe cell surface in association with class I or class II MEC molecules.

Further, the skilled artisan can ascertain an epitope of interest bycomparing the protein sequence with sequences listed in the protein database. Regions of the protein which share little or no homology arebetter choices for being an epitope of that protein and are thereforeuseful in a vaccine or immunological composition. Regions which sharegreat homology with widely found sequences present in vital cells shouldbe avoided.

Even further, another method is simply to generate or express portionsof a protein of interest, generate monoclonal antibodies to thoseportions of the protein of interest, and then ascertain whether thoseantibodies inhibit growth in vitro of the pathogen from which theprotein was derived. The skilled artisan can use the other guidelinesset forth in this disclosure and in the art for generating or expressingportions of a protein of interest for analysis as to whether antibodiesthereto inhibit growth in vitro. For example, the skilled artisan cangenerate portions of a protein of interest by: selecting 8 to 9 or 13 to25 amino acid length portions of the protein, selecting hydrophilicregions, selecting portions shown to bind from X-ray data of the antigen(full length)-antibody complex, selecting regions which differ insequence from other proteins, selecting potential HLA anchor bindingmotifs, or any combination of these methods or other methods known inthe art.

Epitopes recognized by antibodies are expressed on the surface of aprotein. To determine the regions of a protein most likely to stimulatean antibody response one skilled in the art can preferably perform anepitope map, using the general methods described above, or other mappingmethods known in the art.

As can be seen from the foregoing, without undue experimentation, fromthis disclosure and the knowledge in the art, the skilled artisan canascertain the amino acid and corresponding DNA sequence of an epitope ofinterest for obtaining a T cell, B cell and/or antibody response. Inaddition, reference is made to Gefter et al., U.S. Pat. No. 5,019,384,issued May 28, 1991, and the documents it cites, incorporated herein byreference (Note especially the “Relevant Literature” section of thispatent, and column 13 of this patent which discloses that: “A largenumber of epitopes have been defined for a wide variety of organisms ofinterest. Of particular interest are those epitopes to whichneutralizing antibodies are directed.”)

With respect to expression of a biological response modulator, referenceis made to Wohlstadter, “Selection Methods,” WO 93/19170, published Sep.30, 1993, and the documents cited therein, incorporated herein byreference.

For instance, a biological response modulator modulates biologicalactivity; for instance, a biological response modulator is a modulatorycomponent such as a high molecular weight protein associated withnon-NMDA excitatory amino acid receptors and which allostericallyregulates affinity of AMPA binding (See Kendrew, supra). The recombinantof the present invention can express such a high molecular weightprotein.

More generally, nature has provided a number of precedents of biologicalresponse modulators. Modulation of activity may be carried out throughmechanisms as complicated and intricate as allosteric induced quaternarychange to simple presence/absence, e.g., expression/degradation,systems. Indeed, the repression/activation of expression of manybiological molecules is itself mediated by molecules whose activitiesare capable of being modulated through a variety of mechanisms.

Table 2 of Neidhardt et al., Physiology of the Bacterial Cell (SinauerAssociates Inc., Publishers, 1990), at page 73, lists chemicalmodifications to bacterial proteins. As is noted in that table, somemodifications are involved in proper assembly and other modificationsare not, but in either case such modifications are capable of causingmodulation of function. From that table, analogous chemical modulationsfor proteins of other cells can be determined, without undueexperimentation.

In some instances modulation of biological functions may be mediatedsimply through the proper/improper localization of a molecule. Moleculesmay function to provide a growth advantage or disadvantage only if theyare targeted to a particular location. For example, a molecule may betypically not taken up or used by a cell, as a function of that moleculebeing first degraded by the cell by secretion of an enzyme for thatdegradation. Thus, production of the enzyme by a recombinant canregulate use or uptake of the molecule by a cell. Likewise, therecombinant can express a molecule which binds to the enzyme necessaryfor uptake or use of a molecule, thereby similarly regulating its uptakeor use.

Localization targeting of proteins carried out through cleavage ofsignal peptides which is another type of modulation or regulation. Inthis case, a specific endoprotease catalytic activity can be expressedby the recombinant.

Other examples of mechanisms through which modulation of function mayoccur are RNA virus poly-proteins, allosteric effects, and generalcovalent and non-covalent steric hindrance. HIV is a well-studiedexample of an RNA virus which expresses non-functional poly-proteinconstructs. In HIV “the gag, pol, and env poly-proteins are processed toyield, respectively, the viral structural proteins p17, p24, andp15—reverse transcriptase and integrase—and the two envelope proteinsgp41 and gp120” (Kohl et al., PNAS USA 85:4686-90 (1988)). The propercleavage of the poly-proteins is crucial for replication of the virus,and virions carrying inactive mutant HIV protease are non-infectious.This is another example of the fusion of proteins down-modulating theiractivity. Thus, it is possible to construct recombinant viruses whichexpress molecules which interfere with endoproteases, or which provideendoproteases, for inhibiting or enhancing the natural expression ofcertain proteins (by interfering with or enhancing cleavage).

The functional usefulness of enzymes may also be modulated by alteringtheir capability of catalyzing a reaction. Illustrative examples ofmodulated molecules are zymogens, formation/disassociation ofmulti-subunit functional complexes, RNA virus poly-protein chains,allosteric interactions, general steric hindrance (covalent andnon-covalent) and a variety of chemical modifications such asphosphorylation, methylation, acetylation, adenylation, anduridenylation (see Table 1 of Neidhardt, supra, at page 315 and Table 2at page 73).

Zymogens are examples of naturally occurring protein fusions which causemodulation of enzymatic activity. Zymogens are one class of proteinswhich are converted into their active state through limited proteolysis.See Table 3 of Reich, Proteases and Biological Control, Vol. 2, (1975)at page 54). Nature has developed a mechanism of down-modulating theactivity of certain enzymes, such as trypsin, by expressing theseenzymes with additional “leader” peptide sequences at their aminotermini. With the extra peptide sequence the enzyme is in the inactivezymogen state. Upon cleavage of this sequence the zymogen is convertedto its enzymatically active state. The overall reaction rates of thezymogen are “about 10.sup.5-10.sup.6 times lower than those of thecorresponding enzyme” (See Table 3 of Reich, supra at page 54).

It is therefore possible to down-modulate the function of certainenzymes simply by the addition of a peptide sequence to one of itstermini. For example, with knowledge of this property, a recombinant canexpress peptide sequences containing additional amino acids at one orboth termini.

The formation or disassociation of multi-subunit enzymes is another waythrough which modulation may occur. Different mechanisms may beresponsible for the modulation of activity upon formation ordisassociation of multi-subunit enzymes.

Therefore, sterically hindering the proper specific subunit interactionswill down-modulate the catalytic activity. And accordingly, therecombinant of the invention can express a molecule which stericallyhinders a naturally occurring enzyme or enzyme complex, so as tomodulate biological functions.

Certain enzyme inhibitors afford good examples of down-modulationthrough covalent steric hindrance or modification. Suicide substrateswhich irreversibly bind to the active site of an enzyme at acatalytically important amino acid in the active site are examples ofcovalent modifications which sterically block the enzymatic active site.An example of a suicide substrate is TPCK for chymotrypsin (Fritsch,Enzyme Structure and Mechanism, 2d ed; Freeman & Co. Publishers, 1984).This type of modulation is possible by the recombinant expressing asuitable suicide substrate, to thereby modulate biological responses(e.g., by limiting enzyme activity).

There are also examples of non-covalent steric hindrance including manyrepressor molecules. The recombinant can express repressor moleculeswhich are capable of sterically hindering and thus down-modulating thefunction of a DNA sequence by preventing particular DNA-RNA polymeraseinteractions.

Allosteric effects are another way through which modulation is carriedout in some biological systems. Aspartate transcarbamoylase is a wellcharacterized allosteric enzyme. Interacting with the catalytic subunitsare regulatory domains. Upon binding to CTP or UTP the regulatorysubunits are capable of inducing a quaternary structural change in theholoenzyme causing down-modulation of catalytic activity. In contrast,binding of ATP to the regulatory subunits is capable of causingup-modulation of catalytic activity (Fritsch, supra). Using methods ofthe invention, molecules can be expressed which are capable of bindingand causing modulatory quaternary or tertiary changes.

In addition, a variety of chemical modifications, e.g., phosphorylation,methylation, acetylation, adenylation, and uridenylation may be carriedout so as to modulate function. It is known that modifications such asthese play important roles in the regulation of many important cellularcomponents. Table 2 of Neidhardt, supra, at page 73, lists differentbacterial enzymes which undergo such modifications. From that list, oneskilled in the art can ascertain other enzymes of other systems whichundergo the same or similar modifications, without undueexperimentation. In addition, many proteins which are implicated inhuman disease also undergo such chemical modifications. For example,many oncogenes have been found to be modified by phosphorylation or tomodify other proteins through phosphorylation or dephosphorylation.Therefore, the ability afforded by the invention to express modulatorswhich can modify or alter function, e.g., phosphorylation, is ofimportance.

From the foregoing, the skilled artisan can use the present invention toexpress a biological response modulator, without any undueexperimentation.

With respect to expression of fusion proteins by inventive recombinants,reference is made to Sambrook, Fritsch, Maniatis, Molecular Cloning, ALABORATORY MANUAL (2d Edition, Cold Spring Harbor Laboratory Press,1989) (especially Volume 3), and Kendrew, supra, incorporated herein byreference. The teachings of Sambrook et al., can be suitably modified,without undue experimentation, from this disclosure, for the skilledartisan to generate recombinants expressing fusion proteins.

With regard to gene therapy and immunotherapy, reference is made to U.S.Pat. Nos. 4,690,915 and 5,252,479, which are incorporated herein byreference, together with the documents cited therein it and on theirface, and to WO 94/16716 and U.S. application Ser. No. 08/184,009, filedJan. 19, 1994, each of which is also incorporated herein by reference,together with the documents cited therein.

A growth factor can be defined as multifunctional, locally actingintercellular signaling peptides which control both ontogeny andmaintenance of tissue and function (see Kendrew, especially at page 455et seq.).

The growth factor or therapeutic gene, for example, can encode adisease-fighting protein, a molecule for treating cancer, a tumorsuppressor, a cytokine, a tumor associated antigen, or interferon; and,the growth factor or therapeutic gene can, for example, be selected fromthe group consisting of a gene encoding alpha-globin, beta-globin,gamma-globin, granulocyte macrophage-colony stimulating factor, tumornecrosis factor, an interleukin (e.g., an interleukin selected frominterleukins 1 to 14, or 1 to 11, or any combination thereof),macrophage colony stimulating factor, granulocyte colony stimulatingfactor, erythropoietin, mast cell growth factor, tumor suppressor p53,retinoblastoma, interferon, melanoma associated antigen or B7. U.S. Pat.No. 5,252,479 provides a list of proteins which can be expressed in anadenovirus system for gene therapy, and the skilled artisan is directedto that disclosure. WO 94/16716 and U.S. application Ser. No.08/184,009, filed Jan. 19, 1994, provide genes for cytokines and tumorassociated antigens and immunotherapy methods, including ex vivomethods, and the skilled artisan is directed to those disclosures.

Thus, one skilled in the art can create recombinants expressing a growthfactor or therapeutic gene and use the recombinants, from thisdisclosure and the knowledge in the art, without undue experimentation.

Moreover, from the foregoing and the knowledge in the art, no undueexperimentation is required for the skilled artisan to construct aninventive recombinant which expresses an epitope of interest, abiological response modulator, a growth factor, a recognition sequence,a therapeutic gene, or a fusion protein; or for the skilled artisan touse such a recombinant.

It is noted that the exogenous or heterologous DNA can itself include apromoter for driving expression in the recombinant CMV, or the exogenousDNA can simply be coding DNA and appropriately placed downstream from aCMV-endogenous promoter to drive expression. Further, multiple copies ofcoding DNA or use of a strong or early promoter or early and latepromoter, or any combination thereof, can be done so as to amplify orincrease expression. Thus, the exogenous or heterologous DNA can besuitably positioned with respect to a CMV-endogenous promoter, or thosepromoters can be translocated to be inserted at another location, withthe exogenous or heterologous DNA. The coding DNA can be DNA coding formore than one protein so as to have expression of more than one productfrom the recombinant CMV.

The expression products can be antigens, immunogens or epitopes ofinterest; and therefore, the invention further relates to immunological,antigenic or vaccine compositions containing the expression products.Further, since the CMV vector, in certain instances, can be administereddirectly to a suitable host, the invention relates to compositionscontaining the CMV vector. Additionally, since the expression productcan be isolated from the CMV vector in vitro or from cells infected ortransfected by the CMV vector in vitro, the invention relates to methodsfor expressing a product, e.g., which may comprise inserting theexogenous DNA into a CMV as a vector, e.g., by restriction/ligation orby recombination followed by infection or transfection of suitable cellsin vitro with a recombinant CMV, and optionally extracting, purifying orisolating the expression product from the cells. Any suitableextraction, purification or isolation techniques can be employed.

In particular, after infecting cells with the recombinant CMV, theprotein(s) from the expression of the exogenous DNA are collected byknown techniques such as chromatography (see Robbins, EPA 0162738A1;Panicali, EPA 0261940A2); Richardson, supra; Smith et al., supra;Pennock et al., supra; EP Patent Publication No. 0265785). The collectedprotein(s) can then be employed in a vaccine, antigenic or immunologicalcomposition which also contains a suitable carrier.

Thus, the recombinant CMV can be used to prepare proteins such asantigens, immunogens, epitopes of interest, etc. which can be furtherused in immunological, antigenic or vaccine compositions. It is notedthat a recombinant CMV expressing a product detrimental to growth ordevelopment of insects can be used to prepare an insecticide, and arecombinant CMV expressing a product detrimental to growth of plants canbe used to prepare a herbicide (by isolating the expression product andadmixing it with an insecticidally or herbicidally acceptable carrier ordiluent) and a recombinant CMV expressing an anti-fungal polypeptide canbe used to prepare an anti-fungal preparation (by isolating theexpression product and admixing it with a suitable carrier or diluent).

As the expression products can provide an antigenic, immunological orprotective (vaccine) response, the invention further relates to productstherefrom; namely, antibodies and uses thereof. More in particular, theexpression products can elicit antibodies. The antibodies can be formedinto monoclonal antibodies; and, the antibodies or expression productscan be used in kits, assays, tests, and the like involving binding, sothat the invention relates to these uses too. Additionally, since therecombinants of the invention can be used to replicate DNA, theinvention relates to recombinant CMV as a vector and methods forreplicating DNA by infecting or transfecting cells with the recombinantand harvesting DNA therefrom. The resultant DNA can be used as probes orprimers or for amplification.

The administration procedure for recombinant CMV or expression productthereof, compositions of the invention such as immunological, antigenicor vaccine compositions or therapeutic compositions can be via aparenteral route (intradermal, intramuscular, or subcutaneous). Such anadministration enables a systemic immune response. The administrationcan be via a mucosal route, e.g., oral, nasal, genital, etc. Such anadministration enables a local immune response.

More generally, the inventive antigenic, immunological or vaccinecompositions or therapeutic compositions (compositions containing theCMV recombinants of the invention or expression products) can beprepared in accordance with standard techniques well known to thoseskilled in the pharmaceutical arts. Such compositions can beadministered in dosages and by techniques well known to those skilled inthe medical arts taking into consideration such factors as the breed orspecies, age, sex, weight, and condition of the particular patient, andthe route of administration. The compositions can be administered alone,or can be co-administered or sequentially administered with othercompositions of the invention or with other immunological, antigenic orvaccine or therapeutic compositions. Such other compositions can includepurified native antigens or epitopes or antigens or epitopes from theexpression by a recombinant CMV or another vector system; and areadministered taking into account the aforementioned factors.

Examples of compositions of the invention include liquid preparationsfor orifice, e.g., oral, nasal, anal, genital, e.g., vaginal, etc.,administration such as suspensions, syrups or elixirs; and, preparationsfor parenteral, subcutaneous, intradermal, intramuscular or intravenousadministration (e.g., injectable administration) such as sterilesuspensions or emulsions. In such compositions the recombinant may be inadmixture with a suitable carrier, diluent, or excipient such as sterilewater, physiological saline, glucose or the like.

Antigenic, immunological or vaccine compositions typically can containan adjuvant and an amount of the recombinant CMV or expression productto elicit the desired response. In human applications, alum (aluminumphosphate or aluminum hydroxide) is a typical adjuvant. Saponin and itspurified component Quil A, Freund's complete adjuvant and otheradjuvants used in research and veterinary applications have toxicitieswhich limit their potential use in human vaccines. Chemically definedpreparations such as muramyl dipeptide, monophosphoryl lipid A,phospholipid conjugates such as those described by Goodman-Snitkoff etal., J. Immunol. 147:410-415 (1991) and incorporated by referenceherein, encapsulation of the protein within a proteoliposome asdescribed by Miller et al., J. Exp. Med. 176:1739-1744 (1992) andincorporated by reference herein, and encapsulation of the protein inlipid vesicles such as Novasome lipid vesicles (Micro Vescular Systems,Inc., Nashua, N.H.) can also be used.

The composition may be packaged in a single dosage form for immunizationby parenteral (i.e., intramuscular, intradermal or subcutaneous)administration or orifice administration, e.g., perlingual (i.e., oral),intragastric, mucosal including intraoral, intraanal, intravaginal, andthe like administration. And again, the effective dosage and route ofadministration are determined by the nature of the composition, by thenature of the expression product, by expression level if recombinant CMVis directly used, and by known factors, such as breed or species, age,sex, weight, condition and nature of host, as well as LD₅₀ and otherscreening procedures which are known and do not require undueexperimentation. Dosages of expressed product can range from a few to afew hundred micrograms, e.g., 5 to 500 μg. The inventive recombinant canbe administered in any suitable amount to achieve expression at thesedosage levels. The vaccinal CMV is administered in an amount of at least10² pfu; thus, the inventive recombinant can be administered in at leastthis amount; or in a range from about 10² pfu to about 107 pfu. Othersuitable carriers or diluents can be water or a buffered saline, with orwithout a preservative. The expression product or recombinant CMV may belyophilized for resuspension at the time of administration or can be insolution.

The carrier may also be a polymeric delayed release system. Syntheticpolymers are particularly useful in the formulation of a compositionhaving controlled release. An early example of this was thepolymerization of methyl methacrylate into spheres having diameters lessthan one micron to form so-called nanoparticles, reported by Kreuter,J., Microcapsules and Nanoparticles in Medicine and Pharmacology, M.Donbrow (Ed). CRC Press, pp. 125-148.

Microencapsulation has been applied to the injection ofmicroencapsulated pharmaceuticals to give a controlled release. A numberof factors contribute to the selection of a particular polymer formicroencapsulation. The reproducibility of polymer synthesis and themicroencapsulation process, the cost of the microencapsulation materialsand process, the toxicological profile, the requirements for variablerelease kinetics and the physicochemical compatibility of the polymerand the antigens are all factors that must be considered. Examples ofuseful polymers are polycarbonates, polyesters, polyurethanes,polyorthoesters and polyamides, particularly those that arebiodegradable.

A frequent choice of a carrier for pharmaceuticals and more recently forantigens is poly (d,l-lactide-co-glycolide) (PLGA). This is abiodegradable polyester that has a long history of medical use inerodible sutures, bone plates and other temporary prostheses where ithas not exhibited any toxicity. A wide variety of pharmaceuticalsincluding peptides and antigens have been formulated into PLGAmicrocapsules. A body of data has accumulated on the adaption of PLGAfor the controlled release of antigen, for example, as reviewed byEldridge, J. H., et al., Current Topics in Microbiology and Immunology.1989, 146:59-66. The entrapment of antigens in PLGA microspheres of 1 to10 microns in diameter has been shown to have a remarkable adjuvanteffect when administered orally. The PLGA microencapsulation processuses a phase separation of a water-in-oil emulsion. The compound ofinterest is prepared as an aqueous solution and the PLGA is dissolved insuitable organic solvents such as methylene chloride and ethyl acetate.These two immiscible solutions are co-emulsified by high-speed stirring.A non-solvent for the polymer is then added, causing precipitation ofthe polymer around the aqueous droplets to form embryonic microcapsules.The microcapsules are collected, and stabilized with one of anassortment of agents (polyvinyl alcohol (PVA), gelatin, alginates,polyvinylpyrrolidone (PVP), methyl cellulose) and the solvent removed byeither drying in vacuo or solvent extraction.

Thus, solid, including solid-containing-liquid, liquid, and gel(including “gel caps”) compositions are envisioned.

Additionally, the inventive vectors, e.g., recombinant CMV, and theexpression products therefrom can stimulate an immune or antibodyresponse in animals. From those antibodies, by techniques well-known inthe art, monoclonal antibodies can be prepared and, those monoclonalantibodies can be employed in well-known antibody binding assays,diagnostic kits or tests to determine the presence or absence ofantigen(s) and therefrom the presence or absence of the naturalcausative agent of the antigen or, to determine whether an immuneresponse to that agent or to the antigen(s) has simply been stimulated.

Monoclonal antibodies are immunoglobulin produced by hybridoma cells. Amonoclonal antibody reacts with a single antigenic determinant andprovides greater specificity than a conventional, serum-derivedantibody. Furthermore, screening a large number of monoclonal antibodiesmakes it possible to select an individual antibody with desiredspecificity, avidity and isotype. Hybridoma cell lines provide aconstant, inexpensive source of chemically identical antibodies andpreparations of such antibodies can be easily standardized. Methods forproducing monoclonal antibodies are well known to those of ordinaryskill in the art, e.g., Koprowski, H. et al., U.S. Pat. No. 4,196,265,issued Apr. 1, 1989, incorporated herein by reference.

Uses of monoclonal antibodies are known. One such use is in diagnosticmethods, e.g., David, G. and Greene, H., U.S. Pat. No. 4,376,110, issuedMar. 8, 1983, incorporated herein by reference.

Monoclonal antibodies have also been used to recover materials byimmunoadsorption chromatography, e.g. Milstein, C., 1980, ScientificAmerican 243:66, 70, incorporated herein by reference.

Furthermore, the inventive recombinant CMV or expression productstherefrom can be used to stimulate a response in cells in vitro or exvivo for subsequent reinfusion into a patient. If the patient isseronegative, the reinfusion is to stimulate an immune response, e.g.,an immunological or antigenic response such as active immunization. In aseropositive individual, the reinfusion is to stimulate or boost theimmune system against a pathogen.

The recombinant CMV of the invention is also useful for generating DNAfor probes or for PCR primers which can be used to detect the presenceor absence of hybridizable DNA or to amplify DNA, e.g., to detect apathogen in a sample or for amplifying DNA.

Furthermore, as discussed above, the invention comprehends promoters andexpression cassettes which are useful in adenovirus systems, as well asin any viral or cell system which provides a transactivating protein.

The expression cassette of the invention can further include afunctional truncated polyadenylation signal; for instance an SV40polyadenylation signal which is truncated, yet functional. Theexpression cassette can contain exogenous or heterologous DNA (withrespect to the virus or system into which the promoter or expressioncassette is being inserted); for instance exogenous or heterologouscoding DNA as herein described above, and in the Examples. This DNA canbe suitably positioned and operably linked to the promoter forexpression. The expression cassette can be inserted in any orientation;preferably the orientation which obtains maximum expression from thesystem or virus into which the expression cassette is inserted.

While the promoter and expression cassette are specifically exemplifiedwith reference to adenoviruses, the skilled artisan can adapt theseembodiments of the invention to other viruses and to plasmids for cellssuch as eukaryotic cells, without undue experimentation, by simplyascertaining whether the virus, plasmid, cell or system provides thetransactivating protein.

As to HCMV promoters, reference is made to U.S. Pat. Nos. 5,168,062 and5,385,839, incorporated herein by reference. As to transfecting cellswith plasmid DNA for expression therefrom, reference is made to Feigneret al. (1994), J. Biol. Chem. 269, 2550-2561, incorporated herein byreference. And, as to direct injection of plasmid DNA as a simple andeffective method of vaccination against a variety of infectious diseases(reference is made to Science, 259:1745-49, 1993, incorporated herein byreference). It is therefore within the scope of this invention that theinventive promoter and expression cassette be used in systems other thanadenovirus; for example, in plasmids for the direct injection of plasmidDNA.

The protein fragments of the present invention form a further aspect ofthe invention; and, such compounds may be used in methods of medicaltreatments, such as for diagnosis, preventing or treating HIV or foreliciting antibodies for diagnosis of HIV, including use in vaccines.Further, such compounds may be used in the preparation of medicamentsfor such treatments or prevention, or compositions for diagnosticpurposes. The compounds may be employed alone or in combination withother treatments, vaccines or preventatives; and, the compounds may beused in the preparation of combination medicaments for such treatmentsor prevention, or in kits containing the compound and the othertreatment or preventative.

In yet another embodiment, the present invention also encompassed theuse of the protein fragments of the present invention described hereinas immunogens, advantageously as HIV-I vaccine components.

The terms “protein”, “peptide”, “polypeptide”, and “amino acid sequence”are used interchangeably herein to refer to polymers of amino acidresidues of any length. The polymer may be linear or branched, it maycomprise modified amino acids or amino acid analogs, and it may beinterrupted by chemical moieties other than amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling or bioactivecomponent.

As used herein, the terms “antigen” or “immunogen” are usedinterchangeably to refer to a substance, typically a protein, which iscapable of inducing an immune response in a subject. The term alsorefers to proteins that are immunologically active in the sense thatonce administered to a subject (either directly or by administering tothe subject a nucleotide sequence or vector that encodes the protein) isable to evoke an immune response of the humoral and/or cellular typedirected against that protein.

The term “antibody” includes intact molecules as well as fragmentsthereof, such as Fab, F(ab′)2, Fv and scFv which are capable of bindingthe epitope determinant. These antibody fragments retain some ability toselectively bind with its antigen or receptor and include, for example:

-   -   a. Fab, the fragment which contains a monovalent antigen-binding        fragment of an antibody molecule, can be produced by digestion        of whole antibody with the enzyme papain to yield an intact        light chain and a portion of one heavy chain;    -   b. Fab′, the fragment of an antibody molecule, can be obtained        by treating whole antibody with pepsin, followed by reduction,        to yield an intact light chain and a portion of the heavy chain;        two Fab′ fragments are obtained per antibody molecule;    -   c. F(ab′)2, the fragment of the antibody that can be obtained by        treating whole antibody with the enzyme pepsin without        subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragments        held together by two disulfide bonds;    -   d. scFv, including a genetically engineered fragment containing        the variable region of a heavy and a light chain as a fused        single chain molecule.

General methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), which is incorporated herein byreference).

A “neutralizing antibody” may inhibit the entry of HIV-I virus forexample SF162 and/or JRCSF with a neutralization index>1.5 or >2.0.Broad and potent neutralizing antibodies may neutralize greater thanabout 50% of HIV-I viruses (from diverse clades and different strainswithin a clade) in a neutralization assay. The inhibitory concentrationof the monoclonal antibody may be less than about 25 mg/ml to neutralizeabout 50% of the input virus in the neutralization assay.

It should be understood that the proteins and the nucleic acids encodingthem may differ from the exact sequences illustrated and describedherein. Thus, the invention contemplates deletions, additions,truncations, and substitutions to the sequences shown, so long as thesequences function in accordance with the methods of the invention. Inthis regard, substitutions will generally be conservative in nature,i.e., those substitutions that take place within a family of aminoacids. For example, amino acids are generally divided into fourfamilies: (1) acidic—aspartate and glutamate; (2) basic—lysine,arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan; and (4) unchargedpolar—glycine, asparagine, glutamine, cysteine, serine threonine,tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimesclassified as aromatic amino acids. It is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, or viceversa; an aspartate with a glutamate or vice versa; a threonine with aserine or vice versa; or a similar conservative replacement of an aminoacid with a structurally related amino acid, will not have a majoreffect on the biological activity. Proteins having substantially thesame amino acid sequence as the sequences illustrated and described butpossessing minor amino acid substitutions that do not substantiallyaffect the immunogenicity of the protein are, therefore, within thescope of the invention.

As used herein the terms “nucleotide sequences” and “nucleic acidsequences” refer to deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) sequences, including, without limitation, messenger RNA (mRNA),DNA/RNA hybrids, or synthetic nucleic acids. The nucleic acid can besingle-stranded, or partially or completely double-stranded (duplex).Duplex nucleic acids can be homoduplex or heteroduplex.

As used herein the term “transgene” may be used to refer to“recombinant” nucleotide sequences that may be derived from any of thenucleotide sequences encoding the proteins of the present invention. Theterm “recombinant” means a nucleotide sequence that has been manipulated“by man” and which does not occur in nature, or is linked to anothernucleotide sequence or found in a different arrangement in nature. It isunderstood that manipulated “by man” means manipulated by someartificial means, including by use of machines, codon optimization,restriction enzymes, etc.

For example, in one embodiment the nucleotide sequences may be mutatedsuch that the activity of the encoded proteins in vivo is abrogated. Inanother embodiment the nucleotide sequences may be codon optimized, forexample the codons may be optimized for human use. In preferredembodiments the nucleotide sequences of the invention are both mutatedto abrogate the normal in vivo function of the encoded proteins, andcodon optimized for human use. For example, each of the Gag, Pol, Env,Nef, RT, and Int sequences of the invention may be altered in theseways.

As regards codon optimization, the nucleic acid molecules of theinvention have a nucleotide sequence that encodes the antigens of theinvention and can be designed to employ codons that are used in thegenes of the subject in which the antigen is to be produced. Manyviruses, including HIV and other lentiviruses, use a large number ofrare codons and, by altering these codons to correspond to codonscommonly used in the desired subject, enhanced expression of theantigens can be achieved. In a preferred embodiment, the codons used are“humanized” codons, i.e., the codons are those that appear frequently inhighly expressed human genes (Andre et al., J. Virol. 72:1497-1503,1998) instead of those codons that are frequently used by HIV. Suchcodon usage provides for efficient expression of the transgenic HIVproteins in human cells. Any suitable method of codon optimization maybe used. Such methods, and the selection of such methods, are well knownto those of skill in the art. In addition, there are several companiesthat will optimize codons of sequences, such as Geneart (geneart.com).Thus, the nucleotide sequences of the invention can readily be codonoptimized.

The invention further encompasses nucleotide sequences encodingfunctionally and/or antigenically equivalent variants and derivatives ofthe CMV vectors and the glycoproteins included therein. Thesefunctionally equivalent variants, derivatives, and fragments display theability to retain antigenic activity. For instance, changes in a DNAsequence that do not change the encoded amino acid sequence, as well asthose that result in conservative substitutions of amino acid residues,one or a few amino acid deletions or additions, and substitution ofamino acid residues by amino acid analogs are those which will notsignificantly affect properties of the encoded polypeptide. Conservativeamino acid substitutions are glycine/alanine; valine/isoleucine/leucine;asparagine/glutamine; aspartic acid/glutamic acid;serine/threonine/methionine; lysine/arginine; andphenylalanine/tyrosine/tryptophan. In one embodiment, the variants haveat least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% homology or identity to the antigen, epitope,immunogen, peptide or polypeptide of interest.

For the purposes of the present invention, sequence identity or homologyis determined by comparing the sequences when aligned so as to maximizeoverlap and identity while minimizing sequence gaps. In particular,sequence identity may be determined using any of a number ofmathematical algorithms. A nonlimiting example of a mathematicalalgorithm used for comparison of two sequences is the algorithm ofKarlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268,modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993; 90:5873-5877.

Another example of a mathematical algorithm used for comparison ofsequences is the algorithm of Myers & Miller, CABIOS 1988; 4: 11-17.Such an algorithm is incorporated into the ALIGN program (version 2.0)which is part of the GCG sequence alignment software package. Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used. Yet another useful algorithm for identifying regions oflocal sequence similarity and alignment is the FASTA algorithm asdescribed in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85:2444-2448.

Advantageous for use according to the present invention is the WU-BLAST(Washington University BLAST) version 2.0 software. WU-BLAST version 2.0executable programs for several UNIX platforms can be downloaded from.This program is based on WU-BLAST version 1.4, which in turn is based onthe public domain NCBI-BLAST version 1.4 (Altschul & Gish, 1996, Localalignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480;Altschul et al., Journal of Molecular Biology 1990; 215: 403-410; Gish &States, 1993; Nature Genetics 3: 266-272; Karlin & Altschul, 1993; Proc.Natl. Acad. Sci. USA 90: 5873-5877; all of which are incorporated byreference herein).

The various recombinant nucleotide sequences and antibodies and/orantigens of the invention are made using standard recombinant DNA andcloning techniques. Such techniques are well known to those of skill inthe art. See for example, “Molecular Cloning: A Laboratory Manual”,second edition (Sambrook et al. 1989).

The nucleotide sequences of the present invention may be inserted into“vectors.” The term “vector” is widely used and understood by those ofskill in the art, and as used herein the term “vector” is usedconsistent with its meaning to those of skill in the art. For example,the term “vector” is commonly used by those skilled in the art to referto a vehicle that allows or facilitates the transfer of nucleic acidmolecules from one environment to another or that allows or facilitatesthe manipulation of a nucleic acid molecule.

Any vector that allows expression of the viruses of the presentinvention may be used in accordance with the present invention. Incertain embodiments, the viruses of the present invention may be used invitro (such as using cell-free expression systems) and/or in culturedcells grown in vitro in order to produce the encoded HIV-antigens and/orantibodies which may then be used for various applications such as inthe production of proteinaceous vaccines. For such applications, anyvector that allows expression of the virus in vitro and/or in culturedcells may be used.

For the exogenous antigens of the present invention to be expressed, theprotein coding sequence of the exogenous antigen should be “operablylinked” to regulatory or nucleic acid control sequences that directtranscription and translation of the protein. As used herein, a codingsequence and a nucleic acid control sequence or promoter are said to be“operably linked” when they are covalently linked in such a way as toplace the expression or transcription and/or translation of the codingsequence under the influence or control of the nucleic acid controlsequence. The “nucleic acid control sequence” can be any nucleic acidelement, such as, but not limited to promoters, enhancers, IRES,introns, and other elements described herein that direct the expressionof a nucleic acid sequence or coding sequence that is operably linkedthereto. The term “promoter” will be used herein to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II and that when operationally linked to theprotein coding sequences of the invention lead to the expression of theencoded protein. The expression of the transgenes of the presentinvention can be under the control of a constitutive promoter or of aninducible promoter, which initiates transcription only when exposed tosome particular external stimulus, such as, without limitation,antibiotics such as tetracycline, hormones such as ecdysone, or heavymetals. The promoter can also be specific to a particular cell-type,tissue or organ. Many suitable promoters and enhancers are known in theart, and any such suitable promoter or enhancer may be used forexpression of the transgenes of the invention. For example, suitablepromoters and/or enhancers can be selected from the Eukaryotic PromoterDatabase (EPDB).

The present invention relates to a recombinant viral vector expressing aforeign epitope. Advantageously, the epitope is an HIV epitope. In anadvantageous embodiment, the HIV epitope is a protein fragment of thepresent invention, however, the present invention may encompassadditional HIV antigens, epitopes or immunogens. Advantageously, the HIVepitope is an HIV antigen including but not limited to, the HIV antigensof U.S. Pat. Nos. 7,341,731; 7,335,364; 7,329,807; 7,323,553; 7,320,859;7,311,920; 7,306,798; 7,285,646; 7,285,289; 7,285,271; 7,282,364;7,273,695; 7,270,997; 7,262,270; 7,244,819; 7,244,575; 7,232,567;7,232,566; 7,223,844; 7,223,739; 7,223,534; 7,223,368; 7,220,554;7,214,530; 7,211,659; 7,211,432; 7,205,159; 7,198,934; 7,195,768;7,192,555; 7,189,826; 7,189,522; 7,186,507; 7,179,645; 7,175,843;7,172,761; 7,169,550; 7,157,083; 7,153,509; 7,147,862; 7,141,550;7,129,219; 7,122,188; 7,118,859; 7,118,855; 7,118,751; 7,118,742;7,105,655; 7,101,552; 7,097,971; 7,097,842; 7,094,405; 7,091,049;7,090,648; 7,087,377; 7,083,787; 7,070,787; 7,070,781; 7,060,273;7,056,521; 7,056,519; 7,049,136; 7,048,929; 7,033,593; 7,030,094;7,022,326; 7,009,037; 7,008,622; 7,001,759; 6,997,863; 6,995,008;6,979,535; 6,974,574; 6,972,126; 6,969,609; 6,964,769; 6,964,762;6,958,158; 6,956,059; 6,953,689; 6,951,648; 6,946,075; 6,927,031;6,919,319; 6,919,318; 6,919,077; 6,913,752; 6,911,315; 6,908,617;6,908,612; 6,902,743; 6,900,010; 6,893,869; 6,884,785; 6,884,435;6,875,435; 6,867,005; 6,861,234; 6,855,539; 6,841,381; 6,841,345;6,838,477; 6,821,955; 6,818,392; 6,818,222; 6,815,217; 6,815,201;6,812,026; 6,812,025; 6,812,024; 6,808,923; 6,806,055; 6,803,231;6,800,613; 6,800,288; 6,797,811; 6,780,967; 6,780,598; 6,773,920;6,764,682; 6,761,893; 6,753,015; 6,750,005; 6,737,239; 6,737,067;6,730,304; 6,720,310; 6,716,823; 6,713,301; 6,713,070; 6,706,859;6,699,722; 6,699,656; 6,696,291; 6,692,745; 6,670,181; 6,670,115;6,664,406; 6,657,055; 6,657,050; 6,656,471; 6,653,066; 6,649,409;6,649,372; 6,645,732; 6,641,816; 6,635,469; 6,613,530; 6,605,427;6,602,709; 6,602,705; 6,600,023; 6,596,477; 6,596,172; 6,593,103;6,593,079; 6,579,673; 6,576,758; 6,573,245; 6,573,040; 6,569,418;6,569,340; 6,562,800; 6,558,961; 6,551,828; 6,551,824; 6,548,275;6,544,780; 6,544,752; 6,544,728; 6,534,482; 6,534,312; 6,534,064;6,531,572; 6,531,313; 6,525,179; 6,525,028; 6,524,582; 6,521,449;6,518,030; 6,518,015; 6,514,691; 6,514,503; 6,511,845; 6,511,812;6,511,801; 6,509,313; 6,506,384; 6,503,882; 6,495,676; 6,495,526;6,495,347; 6,492,123; 6,489,131; 6,489,129; 6,482,614; 6,479,286;6,479,284; 6,465,634; 6,461,615; 6,458,560; 6,458,527; 6,458,370;6,451,601; 6,451,592; 6,451,323; 6,436,407; 6,432,633; 6,428,970;6,428,952; 6,428,790; 6,420,139; 6,416,997; 6,410,318; 6,410,028;6,410,014; 6,407,221; 6,406,710; 6,403,092; 6,399,295; 6,392,013;6,391,657; 6,384,198; 6,380,170; 6,376,170; 6,372,426; 6,365,187;6,358,739; 6,355,248; 6,355,247; 6,348,450; 6,342,372 6,342,228;6,338,952; 6,337,179; 6,335,183; 6,335,017; 6,331,404; 6,329,202;6,329,173; 6,328,976; 6,322,964; 6,319,666; 6,319,665; 6,319,500;6,319,494; 6,316,205; 6,316,003; 6,309,633; 6,306,625; 6,296,807;6,294,322; 6,291,239; 6,291,157; 6,287,568; 6,284,456; 6,284,194;6,274,337; 6,270,956; 6,270,769; 6,268,484; 6,265,562; 6,265,149;6,262,029; 6,261,762; 6,261,571; 6,261,569; 6,258,599; 6,258,358;6,248,332; 6,245,331; 6,242,461; 6,241,986; 6,235,526; 6,235,466;6,232,120; 6,228,361; 6,221,579; 6,214,862; 6,214,804; 6,210,963;6,210,873; 6,207,185; 6,203,974; 6,197,755; 6,197,531; 6,197,496;6,194,142; 6,190,871; 6,190,666; 6,168,923; 6,156,302; 6,153,408;6,153,393; 6,153,392; 6,153,378; 6,153,377; 6,146,635; 6,146,614;6,143,876; 6,140,059; 6,140,043; 6,139,746; 6,132,992; 6,124,306;6,124,132; 6,121,006; 6,120,990; 6,114,507; 6,114,143; 6,110,466;6,107,020; 6,103,521; 6,100,234; 6,099,848; 6,099,847; 6,096,291;6,093,405; 6,090,392; 6,087,476; 6,083,903; 6,080,846; 6,080,725;6,074,650; 6,074,646; 6,070,126; 6,063,905; 6,063,564; 6,060,256;6,060,064; 6,048,530; 6,045,788; 6,043,347; 6,043,248; 6,042,831;6,037,165; 6,033,672; 6,030,772; 6,030,770; 6,030,618; 6,025,141;6,025,125; 6,020,468; 6,019,979; 6,017,543; 6,017,537; 6,015,694;6,015,661; 6,013,484; 6,013,432; 6,007,838; 6,004,811; 6,004,807;6,004,763; 5,998,132; 5,993,819; 5,989,806; 5,985,926; 5,985,641;5,985,545; 5,981,537; 5,981,505; 5,981,170; 5,976,551; 5,972,339;5,965,371; 5,962,428; 5,962,318; 5,961,979; 5,961,970; 5,958,765;5,958,422; 5,955,647; 5,955,342; 5,951,986; 5,951,975; 5,942,237;5,939,277; 5,939,074; 5,935,580; 5,928,930; 5,928,913; 5,928,644;5,928,642; 5,925,513; 5,922,550; 5,922,325; 5,919,458; 5,916,806;5,916,563; 5,914,395; 5,914,109; 5,912,338; 5,912,176; 5,912,170;5,906,936; 5,895,650; 5,891,623; 5,888,726; 5,885,580; 5,885,578;5,879,685; 5,876,731; 5,876,716; 5,874,226; 5,872,012; 5,871,747;5,869,058; 5,866,694; 5,866,341; 5,866,320; 5,866,319; 5,866,137;5,861,290; 5,858,740; 5,858,647; 5,858,646; 5,858,369; 5,858,368;5,858,366; 5,856,185; 5,854,400; 5,853,736; 5,853,725; 5,853,724;5,852,186; 5,851,829; 5,851,529; 5,849,475; 5,849,288; 5,843,728;5,843,723; 5,843,640; 5,843,635; 5,840,480; 5,837,510; 5,837,250;5,837,242; 5,834,599; 5,834,441; 5,834,429; 5,834,256; 5,830,876;5,830,641; 5,830,475; 5,830,458; 5,830,457; 5,827,749; 5,827,723;5,824,497; 5,824,304; 5,821,047; 5,817,767; 5,817,754; 5,817,637;5,817,470; 5,817,318; 5,814,482; 5,807,707; 5,804,604; 5,804,371;5,800,822; 5,795,955; 5,795,743; 5,795,572; 5,789,388; 5,780,279;5,780,038; 5,776,703; 5,773,260; 5,770,572; 5,766,844; 5,766,842;5,766,625; 5,763,574; 5,763,190 5,762,965 5,759,769 5,756,666 5,753,2585,750,373; 5,747,641; 5,747,526; 5,747,028; 5,736,320; 5,736,146;5,733,760; 5,731,189; 5,728,385; 5,721,095; 5,716,826; 5,716,637;5,716,613; 5,714,374; 5,709,879; 5,709,860; 5,709,843; 5,705,331;5,703,057; 5,702,707; 5,698,178; 5,688,914; 5,686,078; 5,681,831;5,679,784; 5,674,984; 5,672,472; 5,667,964; 5,667,783; 5,665,536;5,665,355; 5,660,990; 5,658,745; 5,658,569; 5,643,756; 5,641,624;5,639,854; 5,639,598; 5,637,677; 5,637,455; 5,633,234; 5,629,153;5,627,025; 5,622,705; 5,614,413; 5,610,035; 5,607,831; 5,606,026;5,601,819; 5,597,688; 5,593,972; 5,591,829; 5,591,823; 5,589,466;5,587,285; 5,585,254; 5,585,250; 5,580,773; 5,580,739; 5,580,563;5,573,916; 5,571,667; 5,569,468; 5,558,865; 5,556,745; 5,550,052;5,543,328; 5,541,100; 5,541,057; 5,534,406; 5,529,765; 5,523,232;5,516,895; 5,514,541; 5,510,264; 5,500,161; 5,480,967; 5,480,966;5,470,701; 5,468,606; 5,462,852; 5,459,127; 5,449,601; 5,447,838;5,447,837; 5,439,809; 5,439,792; 5,418,136; 5,399,501; 5,397,695;5,391,479; 5,384,240; 5,374,519; 5,374,518; 5,374,516; 5,364,933;5,359,046; 5,356,772; 5,354,654; 5,344,755; 5,335,673; 5,332,567;5,320,940; 5,317,009; 5,312,902; 5,304,466; 5,296,347; 5,286,852;5,268,265; 5,264,356; 5,264,342; 5,260,308; 5,256,767; 5,256,561;5,252,556; 5,230,998; 5,230,887; 5,227,159; 5,225,347; 5,221,610;5,217,861; 5,208,321; 5,206,136; 5,198,346; 5,185,147; 5,178,865;5,173,400; 5,173,399; 5,166,050; 5,156,951; 5,135,864; 5,122,446;5,120,662; 5,103,836; 5,100,777; 5,100,662; 5,093,230; 5,077,284;5,070,010; 5,068,174; 5,066,782; 5,055,391; 5,043,262; 5,039,604;5,039,522; 5,030,718; 5,030,555; 5,030,449; 5,019,387; 5,013,556;5,008,183; 5,004,697; 4,997,772; 4,983,529; 4,983,387; 4,965,069;4,945,082; 4,921,787; 4,918,166; 4,900,548; 4,888,290; 4,886,742;4,885,235; 4,870,003; 4,869,903; 4,861,707; 4,853,326; 4,839,288;4,833,072 and 4,795,739.

In another embodiment, HIV, or immunogenic fragments thereof, may beutilized as the HIV epitope. For example, the HIV nucleotides of U.S.Pat. Nos. 7,393,949, 7,374,877, 7,306,901, 7,303,754, 7,173,014,7,122,180, 7,078,516, 7,022,814, 6,974,866, 6,958,211, 6,949,337,6,946,254, 6,896,900, 6,887,977, 6,870,045, 6,803,187, 6,794,129,6,773,915, 6,768,004, 6,706,268, 6,696,291, 6,692,955, 6,656,706,6,649,409, 6,627,442, 6,610,476, 6,602,705, 6,582,920, 6,557,296,6,531,587, 6,531,137, 6,500,623, 6,448,078, 6,429,306, 6,420,545,6,410,013, 6,407,077, 6,395,891, 6,355,789, 6,335,158, 6,323,185,6,316,183, 6,303,293, 6,300,056, 6,277,561, 6,270,975, 6,261,564,6,225,045, 6,222,024, 6,194,391, 6,194,142 6,162,631 6,114,167,6,114,109, 6,090,392, 6,060,587 6,057,102 6,054,565, 6,043,081,6,037,165, 6,034,233, 6,033,902, 6,030,769, 6,020,123, 6,015,661,6,010,895, 6,001,555, 5,985,661, 5,980,900, 5,972,596, 5,939,538,5,912,338, 5,869,339, 5,866,701, 5,866,694, 5,866,320, 5,866,137,5,864,027, 5,861,242, 5,858,785, 5,858,651, 5,849,475, 5,843,638,5,840,480, 5,821,046, 5,801,056, 5,786,177, 5,786,145, 5,773,247,5,770,703, 5,756,674, 5,741,706, 5,705,612, 5,693,752, 5,688,637,5,688,511, 5,684,147, 5,665,577, 5,585,263, 5,578,715, 5,571,712,5,567,603, 5,554,528, 5,545,726, 5,527,895, 5,527,894, 5,223,423,5,204,259, 5,144,019, 5,051,496 and 4,942,122 are useful for the presentinvention.

Any epitope recognized by an HIV antibody may be used in the presentinvention. For example, the anti-HIV antibodies of U.S. Pat. Nos.6,949,337, 6,900,010, 6,821,744, 6,768,004, 6,613,743, 6,534,312,6,511,830, 6,489,131, 6,242,197, 6,114,143, 6,074,646, 6,063,564,6,060,254, 5,919,457, 5,916,806, 5,871,732, 5,824,304, 5,773,247,5,736,320, 5,637,455, 5,587,285, 5,514,541, 5,317,009, 4,983,529,4,886,742, 4,870,003 and 4,795,739 are useful for the present invention.Furthermore, monoclonal anti-HIV antibodies of U.S. Pat. Nos. 7,074,556,7,074,554, 7,070,787, 7,060,273, 7,045,130, 7,033,593, RE39,057,7,008,622, 6,984,721, 6,972,126, 6,949,337, 6,946,465, 6,919,077,6,916,475, 6,911,315, 6,905,680, 6,900,010, 6,825,217, 6,824,975,6,818,392, 6,815,201, 6,812,026, 6,812,024, 6,797,811, 6,768,004,6,703,019, 6,689,118, 6,657,050, 6,608,179, 6,600,023, 6,596,497,6,589,748, 6,569,143, 6,548,275, 6,525,179, 6,524,582, 6,506,384,6,498,006, 6,489,131, 6,465,173, 6,461,612, 6,458,933, 6,432,633,6,410,318, 6,406,701, 6,395,275, 6,391,657, 6,391,635, 6,384,198,6,376,170, 6,372,217, 6,344,545, 6,337,181, 6,329,202, 6,319,665,6,319,500, 6,316,003, 6,312,931, 6,309,880, 6,296,807, 6,291,239,6,261,558, 6,248,514, 6,245,331, 6,242,197, 6,241,986, 6,228,361,6,221,580, 6,190,871, 6,177,253, 6,146,635, 6,146,627, 6,146,614,6,143,876, 6,132,992, 6,124,132, RE36,866, 6,114,143, 6,103,238,6,060,254, 6,039,684, 6,030,772, 6,020,468, 6,013,484, 6,008,044,5,998,132, 5,994,515, 5,993,812, 5,985,545, 5,981,278, 5,958,765,5,939,277, 5,928,930, 5,922,325, 5,919,457, 5,916,806, 5,914,109,5,911,989, 5,906,936, 5,889,158, 5,876,716, 5,874,226, 5,872,012,5,871,732, 5,866,694, 5,854,400, 5,849,583, 5,849,288, 5,840,480,5,840,305, 5,834,599, 5,831,034, 5,827,723, 5,821,047, 5,817,767,5,817,458, 5,804,440, 5,795,572, 5,783,670, 5,776,703, 5,773,225,5,766,944, 5,753,503, 5,750,373, 5,747,641, 5,736,341, 5,731,189,5,707,814, 5,702,707, 5,698,178, 5,695,927, 5,665,536, 5,658,745,5,652,138, 5,645,836, 5,635,345, 5,618,922, 5,610,035, 5,607,847,5,604,092, 5,601,819, 5,597,896, 5,597,688, 5,591,829, 5,558,865,5,514,541, 5,510,264, 5,478,753, 5,374,518, 5,374,516, 5,344,755,5,332,567, 5,300,433, 5,296,347, 5,286,852, 5,264,221, 5,260,308,5,256,561, 5,254,457, 5,230,998, 5,227,159, 5,223,408, 5,217,895,5,180,660, 5,173,399, 5,169,752, 5,166,050, 5,156,951, 5,140,105,5,135,864, 5,120,640, 5,108,904, 5,104,790, 5,049,389, 5,030,718,5,030,555, 5,004,697, 4,983,529, 4,888,290, 4,886,742 and 4,853,326, arealso useful for the present invention.

In one example, the epitope is an SIV epitope. It is understood by oneof skill in the art that anything referring to HIV in the specificationalso applies to SIV. In an advantageous embodiment, the SIV epitope is aprotein fragment of the present invention, however, the presentinvention may encompass additional SIV antigens, epitopes or immunogens.Advantageously, the SIV epitope is an SIV antigen, including but notlimited to, the SIV antigens of U.S. Pat. Nos. 7,892,729; 7,886,962;7,879,914; 7,829,287; 7,794,998; 7,767,455; 7,759,477; 7,758,869;7,754,420; 7,749,973; 7,748,618; 7,732,124; 7,709,606; 7,700,342;7,700,273; 7,625,917; 7,622,124; 7,611,721; 7,608,422; 7,601,518;7,585,675; 7,534,603; 7,511,117; 7,508,781; 7,507,417; 7,479,497;7,464,352; 7,457,973; 7,442,551; 7,439,052; 7,419,829; 7,407,663;7,378,515; 7,364,760; 7,312,065; 7,261,876; 7,220,554; 7,211,240;7,198,935; 7,169,394; 7,098,201; 7,078,516; 7,070,993; 7,048,929;7,034,010; RE39,057; 7,022,814; 7,018,638; 6,955,919; 6,933,377;6,908,617; 6,902,929; 6,846,477; 6,818,442; 6,803,231; 6,800,281;6,797,811; 6,790,657; 6,712,612; 6,706,729; 6,703,394; 6,682,907;6,656,706; 6,645,956; 6,635,472; 6,596,539; 6,589,763; 6,562,571;6,555,523; 6,555,342; 6,541,009; 6,531,574; 6,531,123; 6,503,713;6,479,281; 6,475,718; 6,469,083; 6,468,539; 6,455,265; 6,448,390;6,440,730; 6,423,544; 6,365,150; 6,362,000; 6,326,007; 6,322,969;6,291,664; 6,277,601; 6,261,571; 6,255,312; 6,207,455; 6,194,142;6,117,656; 6,111,087; 6,107,020; 6,080,846; 6,060,064; 6,046,228;6,043,081; 6,027,731; 6,020,123; 6,017,536; 6,004,781; 5,994,515;5,981,259; 5,961,976; 5,950,176; 5,929,222; 5,928,913; 5,912,176;5,888,726; 5,861,243; 5,861,161; 5,858,366; 5,830,475; 5,817,316;5,804,196; 5,786,177; 5,759,768; 5,747,324; 5,705,522; 5,705,331;5,698,446; 5,688,914; 5,688,637; 5,654,195; 5,650,269; 5,631,154;5,582,967; 5,552,269; 5,512,281; 5,508,166; 5,470,572; 5,312,902;5,310,651; 5,268,265; 5,254,457; 5,212,084; 5,087,631 and 4,978,687.

The vectors used in accordance with the present invention shouldtypically be chosen such that they contain a suitable gene regulatoryregion, such as a promoter or enhancer, such that the antigens of theinvention can be expressed.

When the aim is to express antigens of the invention in vivo in asubject, for example in order to generate an immune response against anHIV-1 antigen and/or protective immunity against HIV-1, expressionvectors that are suitable for expression on that subject, and that aresafe for use in vivo, should be chosen. For example, in some embodimentsit may be desired to express the antibodies and/or antigens of theinvention in a laboratory animal, such as for pre-clinical testing ofthe HIV-1 immunogenic compositions and vaccines of the invention. Inother embodiments, it will be desirable to express the antigens of theinvention in human subjects, such as in clinical trials and for actualclinical use of the immunogenic compositions and vaccine of theinvention. Any vectors that are suitable for such uses can be employed,and it is well within the capabilities of the skilled artisan to selecta suitable vector. In some embodiments it may be preferred that thevectors used for these in vivo applications are attenuated to vectorfrom amplifying in the subject. For example, if plasmid vectors areused, preferably they will lack an origin of replication that functionsin the subject so as to enhance safety for in vivo use in the subject.If viral vectors are used, preferably they are attenuated orreplication-defective in the subject, again, so as to enhance safety forin vivo use in the subject.

In preferred embodiments of the present invention viral vectors areused. Advantageously, the vector is a CMV vector, preferably lacking atleast the glycoprotein US11.

In preferred embodiments, the viral vectors of the invention areadministered in vivo, for example where the aim is to produce animmunogenic response in a subject. For example, in some embodiments itmay be desired to express the transgenes of the invention in alaboratory animal, such as for pre-clinical testing of the HIV-1immunogenic compositions and vaccines of the invention. In otherembodiments, it will be desirable to express the antibodies and/orantigens of the invention in human subjects, such as in clinical trialsand for actual clinical use of the immunogenic compositions and vaccineof the invention. In preferred embodiments the subject is a human, forexample a human that is infected with, or is at risk of infection with,HIV-1.

For such in vivo applications the nucleotide sequences, antibodiesand/or antigens of the invention are preferably administered as acomponent of an immunogenic composition which may comprise thenucleotide sequences and/or antigens of the invention in admixture witha pharmaceutically acceptable carrier. The immunogenic compositions ofthe invention are useful to stimulate an immune response against HIV-1and may be used as one or more components of a prophylactic ortherapeutic vaccine against HIV-1 for the prevention, amelioration ortreatment of AIDS. The nucleic acids and vectors of the invention areparticularly useful for providing genetic vaccines, i.e. vaccines fordelivering the nucleic acids encoding the antigens of the invention to asubject, such as a human, such that the antigens are then expressed inthe subject to elicit an immune response.

The compositions of the invention may be injectable suspensions,solutions, sprays, lyophilized powders, syrups, elixirs and the like.Any suitable form of composition may be used. To prepare such acomposition, a nucleic acid or vector of the invention, having thedesired degree of purity, is mixed with one or more pharmaceuticallyacceptable carriers and/or excipients. The carriers and excipients mustbe “acceptable” in the sense of being compatible with the otheringredients of the composition. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include, but are not limited to, water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol, or combinations thereof,buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN® PLURONICS® or polyethylene glycol (PEG).

An immunogenic or immunological composition can also be formulated inthe form of an oil-in-water emulsion. The oil-in-water emulsion can bebased, for example, on light liquid paraffin oil (European Pharmacopeatype); isoprenoid oil such as squalane, squalene, EICOSANE™ ortetratetracontane; oil resulting from the oligomerization of alkene(s),e.g., isobutene or decene; esters of acids or of alcohols containing alinear alkyl group, such as plant oils, ethyl oleate, propylene glycoldi(caprylate/caprate), glyceryl tri(caprylate/caprate) or propyleneglycol dioleate; esters of branched fatty acids or alcohols, e.g.,isostearic acid esters. The oil advantageously is used in combinationwith emulsifiers to form the emulsion. The emulsifiers can be nonionicsurfactants, such as esters of sorbitan, mannide (e.g., anhydromannitololeate), glycerol, polyglycerol, propylene glycol, and oleic,isostearic, ricinoleic, or hydroxystearic acid, which are optionallyethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, suchas the Pluronic® products, e.g., L121. The adjuvant can be a mixture ofemulsifier(s), micelle-forming agent, and oil such as that which iscommercially available under the name Provax® (IDEC Pharmaceuticals, SanDiego, Calif.).

The immunogenic compositions of the invention can contain additionalsubstances, such as wetting or emulsifying agents, buffering agents, oradjuvants to enhance the effectiveness of the vaccines (Remington'sPharmaceutical Sciences, 18th edition, Mack Publishing Company, (ed.)1980).

Adjuvants may also be included. Adjuvants include, but are not limitedto, mineral salts (e.g., AlK(SO₄)₂, AlNa(SO₄)₂, AlNH(SO₄)₂, silica,alum, Al(OH)₃, Ca₃(PO₄)₂, kaolin, or carbon), polynucleotides with orwithout immune stimulating complexes (ISCOMs) (e.g., CpGoligonucleotides, such as those described in Chuang, T. H. et al.,(2002) J. Leuk. Biol. 71(3): 538-44; Ahmad-Nejad, P. et al. (2002) Eur.J. Immunol. 32(7): 1958-68; poly IC or poly AU acids, polyarginine withor without CpG (also known in the art as IC31; see Schellack, C. et al.(2003) Proceedings of the 34th Annual Meeting of the German Society ofImmunology; Lingnau, K. et al. (2002) Vaccine 20(29-30): 3498-508),JuvaVax (U.S. Pat. No. 6,693,086), certain natural substances (e.g., waxD from Mycobacterium tuberculosis, substances found in Cornyebacteriumparvum, Bordetella pertussis, or members of the genus Brucella),flagellin (Toll-like receptor 5 ligand; see McSorley, S. J. et al.(2002) J. Immunol. 169(7): 3914-9), saponins such as QS21, QS17, and QS7(U.S. Pat. Nos. 5,057,540; 5,650,398; 6,524,584; 6,645,495),monophosphoryl lipid A, in particular, 3-de-O-acylated monophosphoryllipid A (3D-MPL), imiquimod (also known in the art as IQM andcommercially available as Aldara®); U.S. Pat. Nos. 4,689,338; 5,238,944;Zuber, A. K. et al. (2004) 22(13-14): 1791-8), and the CCR5 inhibitorCMPD167 (see Veazey, R. S. et al. (2003) J. Exp. Med. 198: 1551-1562).Aluminum hydroxide or phosphate(alum) are commonly used at 0.05 to 0.1%solution in phosphate buffered saline. Other adjuvants that can be used,especially with DNA vaccines, are cholera toxin, especiallyCTA1-DD/ISCOMs (see Mowat, A. M. et al. (2001) J. Immunol. 167(6):3398-405), polyphosphazenes (Allcock, H. R. (1998) App. OrganometallicChem. 12(10-11): 659-666; Payne, L. G. et al. (1995) Pharm. Biotechnol.6: 473-93), cytokines such as, but not limited to, IL-2, IL-4, GM-CSF,IL-12, IL-15 IGF-1, IFN-α, IFN-β, and IFN-γ (Boyer et al., (2002) J.Liposome Res. 121:137-142; WO01/095919), immunoregulatory proteins suchas CD40L (ADX40; see, for example, WO03/063899), and the CD1a ligand ofnatural killer cells (also known as CRONY or α-galactosyl ceramide; seeGreen, T. D. et al., (2003) J. Virol. 77(3): 2046-2055),immunostimulatory fusion proteins such as IL-2 fused to the Fc fragmentof immunoglobulins (Barouch et al., Science 290:486-492, 2000) andco-stimulatory molecules B7.1 and B7.2 (Boyer), all of which can beadministered either as proteins or in the form of DNA, in the same viralvectors as those encoding the antigens of the invention or on separateexpression vectors. Alternatively, vaccines of the invention may beprovided and administered without any adjuvants.

The immunogenic compositions can be designed to introduce the viralvectors to a desired site of action and release it at an appropriate andcontrollable rate. Methods of preparing controlled-release formulationsare known in the art. For example, controlled release preparations canbe produced by the use of polymers to complex or absorb the immunogenand/or immunogenic composition. A controlled-release formulation can beprepared using appropriate macromolecules (for example, polyesters,polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate,methylcellulose, carboxymethylcellulose, or protamine sulfate) known toprovide the desired controlled release characteristics or releaseprofile. Another possible method to control the duration of action by acontrolled-release preparation is to incorporate the active ingredientsinto particles of a polymeric material such as, for example, polyesters,polyamino acids, hydrogels, polylactic acid, polyglycolic acid,copolymers of these acids, or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these active ingredients intopolymeric particles, it is possible to entrap these materials intomicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacrylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed in NewTrends and Developments in Vaccines, Voller et al. (eds.), UniversityPark Press, Baltimore, Md., 1978 and Remington's PharmaceuticalSciences, 16th edition.

Suitable dosages of the viral vectors of the invention (collectively,the immunogens) in the immunogenic composition of the invention can bereadily determined by those of skill in the art. For example, the dosageof the immunogens can vary depending on the route of administration andthe size of the subject. Suitable doses can be determined by those ofskill in the art, for example by measuring the immune response of asubject, such as a laboratory animal, using conventional immunologicaltechniques, and adjusting the dosages as appropriate. Such techniquesfor measuring the immune response of the subject include but are notlimited to, chromium release assays, tetramer binding assays, IFN-γELISPOT assays, IL-2 ELISPOT assays, intracellular cytokine assays, andother immunological detection assays, e.g., as detailed in the text“Antibodies: A Laboratory Manual” by Ed Harlow and David Lane.

The immunogenic compositions can be administered using any suitabledelivery method including, but not limited to, intramuscular,intravenous, intradermal, mucosal, and topical delivery. Such techniquesare well known to those of skill in the art. More specific examples ofdelivery methods are intramuscular injection, intradermal injection, andsubcutaneous injection. However, delivery need not be limited toinjection methods.

Immunization schedules (or regimens) are well known for animals(including humans) and can be readily determined for the particularsubject and immunogenic composition. Hence, the immunogens can beadministered one or more times to the subject. Preferably, there is aset time interval between separate administrations of the immunogeniccomposition. While this interval varies for every subject, typically itranges from 10 days to several weeks, and is often 2, 4, 6 or 8 weeks.For humans, the interval is typically from 2 to 6 weeks. In aparticularly advantageous embodiment of the present invention, theinterval is longer, advantageously about 10 weeks, 12 weeks, 14 weeks,16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30weeks, 32 weeks, 34 weeks, 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44weeks, 46 weeks, 48 weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 58weeks, 60 weeks, 62 weeks, 64 weeks, 66 weeks, 68 weeks or 70 weeks.

The immunization regimes typically have from 1 to 6 administrations ofthe immunogenic composition, but may have as few as one or two or four.The methods of inducing an immune response can also includeadministration of an adjuvant with the immunogens. In some instances,annual, biannual or other long interval (5-10 years) boosterimmunization can supplement the initial immunization protocol.

The present methods also include a variety of prime-boost regimens, forexample DNA prime-Adenovirus boost regimens. In these methods, one ormore priming immunizations are followed by one or more boostingimmunizations. The actual immunogenic composition can be the same ordifferent for each immunization and the type of immunogenic composition(e.g., containing protein or expression vector), the route, andformulation of the immunogens can also be varied. For example, if anexpression vector is used for the priming and boosting steps, it caneither be of the same or different type (e.g., DNA or bacterial or viralexpression vector). One useful prime-boost regimen provides for twopriming immunizations, four weeks apart, followed by two boostingimmunizations at 4 and 8 weeks after the last priming immunization. Itshould also be readily apparent to one of skill in the art that thereare several permutations and combinations that are encompassed using theDNA, bacterial and viral expression vectors of the invention to providepriming and boosting regimens. In the event that the viral vectorsexpress US2-11 or some of the genes encoded in the US2-11 region theycan be used repeatedly while expressing different antigens derived fromdifferent pathogens.

A specific embodiment of the invention provides methods of inducing animmune response against a pathogen in a subject by administering animmunogenic composition of the invention, preferably a CMV vector with adeleterious mutation in at least US11 encoding one or more of theepitopes of the invention, one or more times to a subject wherein theepitopes are expressed at a level sufficient to induce a specific immuneresponse in the subject. Such immunizations can be repeated multipletimes at time intervals of at least 2, 4 or 6 weeks (or more) inaccordance with a desired immunization regime.

The immunogenic compositions of the invention can be administered alone,or can be co-administered, or sequentially administered, with otherantigens, e.g., with “other” immunological, antigenic or vaccine ortherapeutic compositions thereby providing multivalent or “cocktail” orcombination compositions of the invention and methods of employing them.Again, the ingredients and manner (sequential or co-administration) ofadministration, as well as dosages can be determined taking intoconsideration such factors as the age, sex, weight, species andcondition of the particular subject, and the route of administration.

When used in combination, the other antigens can be administered at thesame time or at different times as part of an overall immunizationregime, e.g., as part of a prime-boost regimen or other immunizationprotocol. In an advantageous embodiment, the other HIV immunogen is env,preferably the HIV env trimer.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

EXAMPLES Example 1: Recombinant Cytomegalovirus Vectors with ImprovedImmunogenicity

During the course of evaluation of Rhesus macaque (Rh) CMV/SIV vectorimmunogenicity, SIV epitopes that had been previously shown to representdominant targets of CD8+ T cells in SIV-infected or DNA/Adenovirus/poxvector-vaccinated Rhesus macaques were not targeted at all by RhCMV/SIVvector-elicited CD8+ T cell responses (by ICS or tetramer staining).These included 9 Mamu A*01-restricted epitopes in 12 animals; 3 MamuA*02 epitopes in 4 animals, 1 B*08-epitope in 1 animal, and 3 MamuB*17-epitopes in 7 animals (FIG. 1; left). HCMV and RhCMV express 4related glycoproteins US2/Rh182, US3/Rh184, US6/Rh185 andUS11/Rh189—that act together with very high efficiency to inhibitpresentation of MHC class I-restricted epitopes by infected cells PowersC et al., Curr Top Microbiol Immunol 325, 333-359 (2008); Liu Z et al.,Int J Biochem Cell Biol 41, 503-506 (2009); van der Wal, F J et al.,Curr Top Microbiol Immunol 269, 37-55 (2002); Hewitt E W et al., EMBO J20, 387-396 (2001); all of which are incorporated by reference herein.

The US2-11 region of CMV is shown in FIG. 3. Applicants have generatedone vector that may comprise a deletion encompassing the US2, US3, andUS6 (ΔUS2-6) genes and another that may comprise a deletion of US8,US10, and US11 (ΔUS8-11). Each vector may be generated byBAC-mutagenesis, as described in Hansen S G et al., 2010 supra. Otherconstructs may comprise SIVgag, SIVenv, SIVretanef(rtn), SIVpol, orother exogenous viral, bacterial, parasitic or cancer-derived antigensin place of US2-US6 or US8-11. Additional constructs include individualmutations and/or deletions of US2, US3, US6, US8, US10 or US11 with therest of US2-11 intact. Such constructs may also include exogenousantigens.

Example 2—Construction and Characterization of RhCMVΔUS2-6 andRhCMVΔUS8-11

The vectors Rh186-189 (ΔUS8-11), and Rh182-185 (ΔUS2-6) were generatedthrough BAC recombineering. BAC recombineering begins with recombinationin E. coli between the RhCMV strain 68-1 BAC and a PCR productcontaining the SIV gag or SIVrtn marker and a kanamycin resistant (KanR)cassette. The KanR cassette is flanked by FRT sites, and the ends of thePCR product include between 40-60 base pairs of homology to the ORF tobe deleted. Recombinants are selected with kanamycin, and are thensubjected to arabinose-induced recombination of the FRT sites to deletethe KanR cassette. Therefore, only a gag/rtn marker and a single FRTscar remain in place of the deleted ORF. This final BAC product iselectroporated into rhesus fibroblasts, from which the recombinant virusis harvested. The viruses produced by this method and included in thisstudy are diagrammed in FIGS. 4A and 4B.

All viruses were thoroughly characterized in vitro. All recombinant BACswere screened by restriction digest to demonstrate an intact viralgenome. BACs were also screened by PCR to ensure that the correct ORFswere deleted. Once viruses had been reconstituted from cell culture,their gene expression profiles, SIV protein marker expression, andgrowth kinetics were assayed. Semi-quantitative RT-PCR confirmed thatthe knockout strategy had deleted the appropriate ORFs without affectingsurrounding transcripts or cellular controls GAPDH or β-actin (FIG. 5A).In addition, Western blot of infected cell lysate confirmed expressionof either SIVgag or SIVrtn (tagged with Flag or V5, respectively). Allinfected cell lysates expressed viral protein IE-I or IE-2 (FIG. 5B).

RhCMV lacking homologues of HCMV US8-11 causes superinfection andelicits gag-specific immunodominant responses. Applicants infected twoMamu A*O1 RhCMV-seropositive rhesus macaques (RM) with a viruscontaining a targeted deletion within the Rh182-189 region that lackedthe ORFs Rh186-Rh189 (corresponding to HCMV US8-11) but contained theexogenous antigen SIVgag driven by the EF1α promoter (ΔUS8-11gag) (FIGS.4A and 4B). This virus still contains the majority of the MHC-Iinhibitors, including homologues to HCMV US2, US3, and US6. TheΔUS8-11gag was able to overcome preexisting immunity to RhCMV andsuperinfect both Mamu A*01 RM, as determined by multiparameter flowcytometry of PBMCs and BAL collected from the two animals (FIG. 6A). Inaddition, both animals developed SIVgag-specific PBMC and BAL CD4+ andCD8+ T cells responses within 2 weeks of ΔUS8-11gag inoculation (FIG.6B). The total SIVgag-specific T cell responses were measured by using apool of overlapping peptides. Strikingly, both RM developed the sameMamu A*01-restricted SIVgag immunodominant responses seen withΔUS2-11gag (FIGS. 1 and 6C). These data show that US8-11-deleted vectorscan super-infect but also induce T cells to immunodominant epitopes.

Example 3—CMV Vectors Lacking US8-11 are Able to Super-InfectCMV-Positive Rhesus Macaques (RM) and CMV/SIV Vectors Lacking US8-11Induce a Long-Term CD8+ T Cell Response to Typical Immune-Dominant SIVEpitopes

Four CMV-positive RM were inoculated subcutaneously with 107plaque-forming units (PFU) of recombinant ΔUS8-11RhCMV/rtn andΔUS8-11RhCMV/gag vector. Blood or BAL was collected at the indicateddays and T cell responses were analyzed on the same day. In FIG. 7A,CD8+ T cell responses frequencies to the SIV antigens SIVgag and SIVrtn(fusion of rev-tat-nef) determined by flow cytometric analysis ofintracellular cytokine staining for CD8+ T cells and the activationmarkers CD69, TNF-α and IFN-γ after stimulation of PBMC with overlappingpeptides covering the SIV antigens. The percentage of the responding,SIVrtn or SIVgag-specific T cells within the overall memory subset inboth the blood (left) and BAL (right) fractions are shown for each timepoint as the mean for all four RM (+/−SEM). The development andpersistence of T cell responses against SIVrtn and SIVgag indicates theability of US8-11-deleted vectors to super-infect CMV+RM. In FIG. 7B,CD8+ T cell responses frequencies to the immunodominant MamuA*OI-restricted epitopes SIVtat(SL8) and SIVgag(CM9) determined by flowcytometric analysis of intracellular cytokine staining for CD8+ T cellsand the activation markers CD69, TNF-α and IFN-γ after stimulation ofPBMC with SL8 and CM9 9-mer peptides. The percentage of the responding,SIVtat(SL8) or SIVgag(CM9) specific T cells within the overall memorysubset in both the blood (left) and BAL (right) fractions are shown foreach time point as the mean for all four RM (+/−SEM). The development ofT cell responses against immunodominant epitopes tatSL8 and gagCM9indicates the ability of US8-11-deleted vectors to elicit CD8+ T cellresponses to immunodominant epitopes that are not targeted for CD8+ Tcell responses by wildtype RhCMVrtn- or RhCMgag-expressing vectors.

Example 4—CMV Vectors Lacking US2-6 are Able to Super-InfectCMV-Positive Rhesus Macaques (RM) but do not Induce a CD8+ T CellResponse to Typical Immune-Dominant SIV Epitopes

Four CMV-positive RM were inoculated subcutaneously with 10⁷plaque-forming units (PFU) of recombinant ΔUS2-6RhCMV/rtn andΔUS2-6RhCMV/gag vector. Blood or BAL was collected at the indicated daysand T cell responses were analyzed on the same day. In FIG. 8A, CD8+ Tcell responses frequencies to the SIV antigens SIVgag and SIVrtn (fusionof rev-tat-nef) determined by flow cytometric analysis of intracellularcytokine staining for CD8+ T cells and the activation markers CD69,TNF-α and IFN-γ after stimulation of PBMC with overlapping peptidescovering the SIV antigens. The percentage of the responding, SIVrtn orSIVgag-specific T cells within the overall memory subset in the blood(left) and BAL (right) fractions are shown for each time point as themean for all four RM (+/−SEM). The development and persistence of T cellresponses against SIVrtn and SIVgag indicates the ability ofUS2-6-deleted vectors to super-infect CMV+RM. In FIG. 7B, CD8+ T cellresponses frequencies to the immunodominant Mamu A*01-restrictedepitopes SIVtat(SL8) and SIVgag(CM9) determined by flow cytometricanalysis of intracellular cytokine staining for CD8+ T cells and theactivation markers CD69, TNF-α and IFN-γ after stimulation of PBMC withSL8 and CM9 9-mer peptides. The percentage of the responding,SIVtat(SL8) or SIVgag(CM9) specific T cells within the overall memorysubset in the blood (left) and BAL (right) fractions are shown for eachtime point as the mean for all four RM (+/−SEM). The lack of T cellresponses against immunodominant epitopes tatSL8 and gagCM9 indicatesthat US2-6-deleted vectors are unable to induce CD8+ T cell responses toimmunodominant epitopes similar to wildtype RhCMVrtn- orRhCMgag-expressing vectors.

Example 5—Deletion of Rh189(US11) by Gag-Insertion in RhCMV-Retanef

FIG. 9A shows a schematic representation of the construct RTNΔ189gag.The inhibitor of antigen presentation Rh189 (US11) was deleted byinsertion of a promoterless SIVgag. SIVretanef was inserted betweenRh213 and 214 and is driven by the EF1α promoter as described (Hansen etal. Nat. Med. 2009).

FIG. 9B shows a verification of Rh189-deletion and SIVgag insertion bypolymerase chain reaction. Lysates of rhesus fibroblasts uninfected orinfected with the indicated viruses were subjected to PCR using primersspecific for the indicated inserts. Note that construct RTNΔRh189gagdoes not yield a Rh189-specific DNA fragment, only non-specific bandsalso found in uninfected cells. In contrast, probing for SIVgag or forthe neighboring open reading frame Rh190 results in a specific PCRproduct.

FIG. 9C shows an Immunoblot for SIVretanef. Lysates of fibroblastsinfected with the indicated viruses were separated by SDS-PAGE and aftertransfer onto immunoblot membranes probed with an antibody against theV5-epitope that is fused to the rev-tat-nef (rtn) fusion protein of SIV.Note that only in viruses expressing SIVrtn the respective protein isdetectable.

Example 6—FIG. 10: RhCMV Lacking Rh189(US11) is Able to Super-InfectCMV+ Animals and Induces an Immune Response Against Immunodominant SIVEpitopes

A CMV-positive RM was inoculated subcutaneously with 10⁷ plaque-formingunits (PFU) of recombinant RhCMV/RTNΔ189gag. The Figure shows CD8+ Tcell responses frequencies to overlapping peptides of SIVrtn a fusion ofrev/tat and nef or against the immunodominant Mamu A*01-restrictedepitope SL8 of SIVtat as determined by flow cytometric analysis ofintracellular cytokine staining for CD8+ T cells and the activationmarkers TNF-α and IFN-γ after stimulation of peripheral blood (toppanels) and BAL T cells (bottom panels) with peptides. Depicted are Tcells from a representative RM responding to SIVrtn (left panels) orSIVtat(SL8) (right panels). The upper and lower right quadrants of theflow cytometric profiles indicate the net percentage of the total CD8+ Tcell population responding to the designated antigen with production ofboth TNF and IFN-γ or TNF alone, respectively.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

SEQUENCE LISTING US2 Protein-SEQ ID NO: 1   1mnnlwkawvg lwtsmgplir lpdgitkage dalrpwksta khpwfqiedn rcyidngklf  61argsivgnms rfvfdpkady ggvgenlyvh addvefvpge slkwnvrnld vmpifetlal 121rlvlqgdviw lrcvpelrvd ytssaymwnm qygmvrksyt hvawtivfys initllvlfi 181vyvtvdcnls mmwmrffvc US3 Protein-SEQ ID NO: 2   1mkpvlvlail avlflrlads vprpldvvvs eirsahfrve enqcwfhmgm lhykgrmsgn  61ftekhfvsvg ivsqsymdrl qvsgeqyhhd ergayfewni gghpvphtvd mvditlstrw 121gdpkkyaacv pqvrmdyssq tinwylqrsi rddnwgllfr tllvylfslv vlvlltvgvs 181arlrfi US6 Protein-SEQ ID NO: 3   1mdllirlgfl lmcalptpge rssrdpitll slsprqqacv prtksyrpvc yndtgdctda  61ddswkqlsed fahqclqaak krpkthksrp ndrnlegrlt cqrvsrllpc dldihpshrl 121ltlmndcvcd gavwnafrli erhgffavtl ylccgitllv vilallcsit yestgrgirr 181cgs US11 Protein-SEQ ID NO: 4   1mnlvmlilal wapvagsmpe lsltlfdepp plveteplpp lpdvseyrve ssearcvlrs  61ggrlealwtl rgnlsvptpt prvyyqtleg yadrvptpve dvseslvakr ywlrdyrvpq 121rtklvlfyfs pchqcqtyyv eceprclvpw vplwssledi erllfedrrl mayyaltiks 181aqytlmmvav iqvfwglyvk gwlhrhfpwm fsdqw

What is claimed is:
 1. A cytomegalovirus (CMV) vector comprising: a first nucleic acid sequence encoding US2, US3, or US6 or a homolog thereof; and a second nucleic acid sequence encoding an exogenous viral protein, wherein the exogenous viral protein is an Adenovirus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, Herpes simplex, type 1, Herpes simplex, type 2, Varicella-zoster virus, Epstein-barr virus, Kaposi's sarcoma herpesvirus, Hepatitis B virus, Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, Human immunodeficiency virus (HIV), Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Human metapneumovirus, Human papillomavirus, Rabies virus, Rubella virus, Human bocavirus, or Parvovirus B19 protein; wherein the vector does not encode a functional US11 protein, or a homolog thereof.
 2. The vector of claim 1, wherein the first nucleic acid sequence encodes US2, US3, and US6.
 3. The vector of claim 2, wherein a nucleic acid encoding a US11 ORF is deleted.
 4. The vector of claim 1, further comprising a third nucleic acid sequence encoding US11 and wherein the nucleic acid sequence encoding US11 comprises a point mutation, a frameshift mutation, and/or a deletion of one or more nucleotides of the nucleic acid sequence encoding US11.
 5. The vector of claim 1, wherein the exogenous viral protein is a Hepatitis B protein.
 6. The vector of claim 5, wherein the exogenous viral protein is HIVgag, HIVenv, HIVrev, HIVtat, HIVnef, HIVpol, HIVint, SIVgag, SIVenv,, SIVrev SIVtat, SIVnef, or SIVpol.
 7. A method of eliciting an immune response to an antigen in a subject, the method comprising administering an effective amount of a cytomegalovirus (CMV) vector according to claim
 1. 8. The method of claim 7, wherein the subject was previously exposed to CMV.
 9. The method of claim 7, wherein the CMV vector comprises one or more of a point mutation in a nucleic acid sequence encoding US11, a frameshift mutation in the nucleic acid sequence encoding US11, a deletion of all or part of the nucleic acid sequence encoding US11, or an antisense or RNAi construct that inhibits the expression of US11.
 10. The method of claim 9, wherein the subject is a human or a rhesus macaque.
 11. The method of claim 7, wherein administering comprises intravenous, intramuscular, intraperitoneal, or oral administration of the CMV vector.
 12. The vector of claim 1, wherein the first nucleic acid sequence encodes a US2 comprising the amino acid sequence of SEQ ID NO: 1, a US3 comprising the amino acid sequence of SEQ ID NO: 2, or a US6 comprising the amino acid sequence of SEQ ID NO:3.
 13. The vector of claim 1, wherein the first nucleic acid sequence encodes a US2 comprising the amino acid sequence of SEQ ID NO: 1, a US3 comprising the amino acid sequence of SEQ ID NO:2, and a US6 comprising the amino acid sequence of SEQ ID NO:
 3. 14. The vector of claim 1, wherein the functional US11 protein comprises the amino acid sequence of SEQ ID NO:
 4. 15. The vector of claim 2, wherein the functional US11 protein comprises the amino acid sequence of SEQ ID NO:4.
 16. The vector of claim 3, wherein the functional US11 protein comprises the amino acid sequence of SEQ ID NO:
 4. 17. The vector of claim 1, wherein the vector does not encode a functional US8 or a functional US10 protein, or homolog thereof.
 18. The vector of claim 17, wherein the vector comprises a deletion of all of the nucleic acid sequence encoding US8-US11. 